Factor VIIa inhibitor

ABSTRACT

The present invention relates to novel inhibitors of Factors VIIa, IXa, Xa, XIa, in particular Factor VIIa, pharmaceutical compositions comprising these inhibitors, and methods for using these inhibitors for treating or preventing thromboembolic disorders, cancer or rheumatoid arthritis. Processes for preparing these inhibitors are also disclosed.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.13/623,578, filed Sep. 20, 2012, which is a continuation of U.S.application Ser. No. 13/100,058, filed May 3, 2011, now issued as U.S.Pat. No. 8,415,328 on Apr. 9, 2013, which is a continuation of U.S.application Ser. No. 11/597,335, filed Jul. 14, 2008, which is theNational Phase entry of International Application No. PCT/US2005/019394,filed Jun. 2, 2005, which claims the benefit of U.S. ProvisionalApplication No. 60/576,382, filed Jun. 2, 2004, all of which are herebyincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to novel inhibitors of Factor VIIa,pharmaceutical compositions comprising these inhibitors, and methods forusing these inhibitors for treating or preventing disorders mediated byFactor VIIa. Processes for preparing these inhibitors are alsodisclosed.

2. State of the Art

Thrombosis results from a complex sequence of biochemical events, knownas the coagulation cascade. A triggering event in coagulation is thebinding of the serine protease Factor VIIa (FVIIa), found in thecirculation, to tissue factor (TF), a receptor, which is found on thesurface of blood vessels after damage or inflammation. Once bound to TF,Factor VIIa catalyzes the formation of the serine protease Factor Xa,which subsequently forms the final protease in the cascade, thrombin.

The clinical manifestations of thrombosis range from acute myocardialinfarction (AMI or heart attack) and unstable angina (UA), which occurin the key blood vessels of the heart (coronary vasculature) to deepvein thrombosis (DVT), which is the formation of blood clots in lowerextremities and which often follows orthopedic surgery on the hip andknee, as well as general abdominal surgery and paralysis. Formation ofDVT is a risk factor for the development of pulmonary embolism (PE) inwhich part of a blood clot formed in the lower extremities breaks offand travels to the lung where it blocks the flow of blood. Theunpredictable development of PE often leads to a fatal outcome.Thrombosis can also be generalized systemically, with microclotformation occurring throughout the vascular system. This condition,known as disseminated intravascular coagulation (DIC), can be aconsequence of certain viral diseases such as Ebola, certain cancers,sepsis, and rheumatoid arthritis. Severe DIC can lead to a dramaticreduction in the coagulation factors due to the excessive activation ofthe clotting response that may result in multiple organ failure,hemorrhage, and death.

The formation or embolization of blood clots in the blood vessels of thebrain is the key event resulting in ischemic stroke. Triggering factorsthat lead to stroke are atrial fibrillation or abnormal rhythm of theatria of the heart and atherosclerosis followed by thrombosis in themain artery leading from the heart to the brain (carotid artery). Over600,000 individuals suffer strokes each year in the U.S. Two-thirds ofthese stroke victims suffer some disability, and one-third sufferpermanent and severe disability. Accordingly, there is a need forantithrombotic agents for the treatment of a variety of thromboticconditions. The present invention fulfills this and related needs.

SUMMARY OF THE INVENTION

In one aspect this invention is directed to a compound of Formula I:

wherein:

X¹, X², X³, and X⁴ are independently —N— or —CR⁴— wherein R⁴ ishydrogen, alkyl, or halo with the proviso that not more than three ofX¹, X², X³ and X⁴ are —N—;

R¹ is hydrogen, alkyl, halo, carboxy or aminocarbonyl;

R² is hydrogen, alkyl, or halo;

R³ is dicarboxyalkylaminocarbonylalkyl ordicarboxyalkylaminocarbonylcycloalkyl;

R^(x) is hydrogen, alkyl, alkylthio, halo, hydroxy, hydroxyalkyl,alkoxy, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, ornitro;

R^(y) is hydrogen, alkyl, or halo;

R^(z) is hydrogen, alkyl, haloalkyl, cycloalkyl, alkylthio, halo,hydroxy, hydroxyalkyl, nitro, cyano, alkoxy, alkoxyalkyl,alkoxyalkyloxy, hydroxyalkyloxy, aminoalkyloxy, carboxyalkyloxy,aminocarbonylalkyloxy, haloalkoxy, carboxy, carboxyalkyl,alkoxycarbonyl, alkoxycarbonylalkyl, cyanoalkyl, alkylsulfonyl,alkylsulfonylalkyl, arylsulfonyl, heteroarylsulfonyl, carbamimidoyl,hydroxycarbamimidoyl, alkoxycarbamimidoyl, alkylsulfonylamino,alkylsulfonylaminoalkyl, alkoxysulfonylamino, alkoxysulfonylaminoalkyl,heterocycloalkylalkylaminocarbonyl, hydroxyalkoxyalkylaminocarbonyl,heterocycloalkylcarbonyl, heterocycloalkylcarbonylalkyl,heterocycloalkyl, heterocycloalkylalkyl, oxoheterocycloalkyl,oxoheterocycloalkylalkyl, heteroaryl, heteroaralkyl, ureido,alkylureido, dialkylureido, ureidoalkyl, alkylureidoalkyl,dialkylureidoalkyl, thioureido, thioureidoalkyl, —COR¹² (where R¹² isalkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, or aminoalkyl),-(alkylene)-COR¹² (where R¹² is alkyl, haloalkyl, hydroxyalkyl,alkoxyalkyl, or aminoalkyl), —CONR¹⁴R¹⁵ (where R¹⁴ is hydrogen or alkyland R¹⁵ is hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, or heteroaralkyl), -(alkylene)-CONR¹⁶R¹⁷ (where R¹⁶ ishydrogen, alkyl or hydroxyalkyl and R¹⁷ is hydrogen, alkyl,hydroxyalkyl, alkoxyalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl),—NR¹⁸R¹⁹ (where R¹⁸ is hydrogen or alkyl and R¹⁹ is hydrogen, alkyl,acyl, aryl, aralkyl, heteroaryl, or heteroaralkyl), -(alkylene)-NR²⁰R²¹(where R²⁰ is hydrogen, alkyl, or hydroxyalkyl and R²¹ is hydrogen,alkyl, acyl, alkoxycarbonyl, hydroxyalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, or heteroaralkyl), —SO₂NR²²R²³ (where R²² is hydrogen oralkyl and R²³ is hydrogen, alkyl, aryl, aralkyl, heteroaryl, orheteroaralkyl, or R²² and R²³ together with the nitrogen atom to whichthey are attached form heterocycloamino), -(alkylene)-SO₂NR²⁴R²⁵ (whereR²⁴ is hydrogen or alkyl and R²⁵ is hydrogen, alkyl, aryl, aralkyl,heteroaryl, or heteroaralkyl or R²⁴ and R²⁵ together with the nitrogenatom to which they are attached form heterocycloamino), —NR²⁶SO₂NR²⁷R²⁸(where R²⁶ and R²⁷ are independently hydrogen or alkyl, and R²⁸ ishydrogen, alkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl or R²⁷ andR²⁸ together with the nitrogen atom to which they are attached formheterocycloamino), -(alkylene)-NR²⁹SO₂NR³⁰R³¹ (where R²⁹ and R³⁰ areindependently hydrogen or alkyl, and R³¹ is hydrogen, alkyl, aryl,aralkyl, heteroaryl, or heteroaralkyl or R³⁰ and R³¹ together with thenitrogen atom to which they are attached form heterocycloamino),—CONH-(alkylene)-NR³²R³³ where R³² is hydrogen or alkyl and R³³ isalkyl), or aralkyl; and

R¹³ is hydrogen, hydroxy, (C₁₋₁₀)alkoxy, —C(O)R³⁵ where R³⁵ is alkyl,aryl, haloalkyl, or cyanoalkyl, or —C(O)OR³⁶ where R³⁶ is alkyl,hydroxyalkyl, alkoxyalkyl, alkoxycarbonylalkyl, acyl, aryl, orhaloalkyl; or

a zwitterions thereof; or a pharmaceutically acceptable salt thereofprovided that the compound of Formula I is not(RS)-2-{2-[5-(5-carbamimIdoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinicacid;

(RS)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]-2-methylpropionylamino}succinicacid; and

(RS)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-ureidomethyllbiphenyl-3-yl]-2-methylpropionylamino}succinicacid or a pharmaceutically acceptable salt thereof. The names of thecompounds were generated using AutoNom version 2.2.

In a second aspect, this invention is directed to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of a compound of Formula I, or azwitterion thereof, or a pharmaceutically acceptable salt thereof.

In a third aspect, this invention is directed to a method of treating adisease in an animal that is mediated by Factors VIIa, IXa, Xa and/orXIa, preferably VIIa, which method comprises administering to saidanimal a therapeutically effective amount of a compound of Formula I, ora zwitterions thereof, or a pharmaceutically acceptable salt thereof.Preferably, the disorder is a thromboembolic disorder or cancer orrheumatoid arthritis, more preferably a thromboembolic disorder, evenmore preferably the disorder is deep vein thrombosis. Preferably, thecompound of the invention is administered prophylactically.

In a fourth aspect, this invention is directed to a method of treating athromboembolic disorder in an animal which method comprisesadministering to said animal a therapeutically effective amount of acompound of Formula I, or a zwitterion thereof, or a pharmaceuticallyacceptable salt thereof in combination with another anticoagulantagent(s) independently selected from a group consisting of a thrombininhibitor, factor IXa inhibitor, factor Xa inhibitor, Aspirin®, andPlavix®.

In a fifth aspect, this invention is directed to a method for inhibitingthe coagulation of a biological sample (e.g., stored blood products andsamples) comprising the administration of a compound of Formula I, or azwitterion thereof, or a pharmaceutically acceptable salt thereof.

In a sixth aspect, this invention directed to the use of a compound ofFormula I, or a zwitterion thereof, or a pharmaceutically acceptablesalt thereof in the preparation of a medicament. The medicament isuseful in the treatment of a thromboembolic disorder or cancer orrheumatoid arthritis in an animal. Preferably, the disorder is athromboembolic disorder such as deep vein thrombosis.

In a seventh aspect, this invention is directed to an intermediate offormula (II):

where PG¹ is a suitable oxygen-protecting group and PG² is a suitableamino-protecting group. Preferably, PG¹ is alkyl or aralkyl, morepreferably benzyl. Preferably, PG² is alkyl, more preferably tert-butyl.

In an eighth aspect, this invention is directed to a process ofpreparing a compound of Formula I where R³ isdicarboxyalkylaminocarbonylalkyl comprising:

(i) reacting a compound of formula:

where R¹, R², R¹³, R^(x), R^(y), R^(z), X¹-X⁴ are as defined in theSummary of the Invention or a suitably protected derivative thereof;with dicarboxyalkylamino where the carboxy groups are optionallyprotected;

(ii) optionally removing the any protecting groups;

(iii) optionally modifying any of the R¹, R², R¹³, R^(x), R^(y), andR^(z) groups;

(iii) optionally converting the product from step (ii) or (iii) above,to an acid addition salt;

(iv) optionally converting the product from step (ii) or (iii) above, toa free base;

(v) optionally converting the product from step (ii) or (iii) above, toa zwitterions.

In a ninth aspect, this invention is directed to a process of preparinga compound of Formula Ib or Ic:

comprising:

-   -   (i) reacting a compound of formula:

-   -   with (R) or (S) aspartic acid respectively, where the carboxy        groups are optionally protected;    -   (ii) optionally deprotecting any protected carboxy group(s);    -   (ii) optionally converting the product from step (i) or (ii)        above, to an acid addition salt;    -   (iii) optionally converting the product from step (i) or (ii)        above, to a free base;    -   (iv) optionally converting the product from step (i) or (ii)        above, to a zwitterion;

optionally deprotecting any protected carboxy group(s).

Preferably, the carboxy groups are protected with benzyl groups and thecompound being prepared is Ib.

In a tenth aspect, this invention is directed to a method of preparingan intermediate of formula (II):

where PG¹ is a suitable oxygen-protecting group and PG² is a suitableamino-protecting group comprising:(i) reacting a compound of formula (III):

where PG¹ and PG² is are as defined above and X is halo; with oneequivalent of an organometallic agent of formula RLi or RMgX¹ where R isalkyl or aryl and X¹ is halo to deprotonate the —SO₂NHPG² group;(ii) transmetallating the compound generated in Step (i) above with oneequivalent of an organometallic agent of formula RLi or RMgX¹ where R isalkyl or aryl;(iii) treating the compound generated in Step (ii) above withtrialkylborate to generate a compound of formula (II).

Preferably, methylmagnesium bromide is used in Step (i) above;i-propylmagnesium bromide is used in Step (ii) above and thetrialkylborate is trimethylborate. Preferably, in compound (III) above,PG¹ is benzyl, PG² is tert-butyl and X is iodo.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms, as used in the present specification and claims,are intended to have the meanings as defined below, unless indicatedotherwise or used in naming a compound.

“Alkyl” means a linear saturated monovalent hydrocarbon radical of oneto six carbon atoms or a branched saturated monovalent hydrocarbonradical of three to six carbon atoms, e.g., methyl, ethyl, propyl,2-propyl, butyl (including all isomeric forms), pentyl (including allisomeric forms), and the like.

“Alkylene” means a linear saturated divalent hydrocarbon radical of oneto six carbon atoms or a branched saturated divalent hydrocarbon radicalof three to six carbon atoms e.g., methylene, ethylene, propylene,1-methylpropylene, 2-methylpropylene, butylene, pentylene, and the like.

“Alkylthio” means a radical —SR where R is alkyl as defined above, e.g.,methylthio, ethylthio, propylthio (including all isomeric forms),butylthio (including all isomeric forms), and the like.

“Amino” means the radical —NRR′ where R and R′ are independentlyhydrogen, alkyl, or —COR^(a) where R^(a) is alkyl, e.g., —NH₂,methylaminoethyl, 1,3-diaminopropyl, acetylaminopropyl, and the like.

“Alkylamino” means a radical —NHR where R is alkyl as defined above,e.g., methylamino, ethylamino, propylamino (including all isomericforms), and the like.

“Acyl” means a radical —COR′ where R′ is alkyl, alkoxy, haloalkyl,aminoalkyl, hydroxyalkyl, or alkoxyalkyl as defined herein, e.g.,acetyl, trifluoroacetyl, hydroxymethylcarbonyl, and the like.

“Aminosulfonyl” or “sulfamoyl” means a radical —SO₂NH₂.

“Alkylaminosulfonyl” means a radical —SO₂NHR where R is alkyl as definedabove, e.g., methylaminosulfonyl, ethylamino-sulfonyl, and the like.

“Alkylsulfonyl” means a radical —SO₂R where R is alkyl as defined above,e.g., methylsulfonyl, ethylsulfonyl, n- or iso-propylsulfonyl, and thelike.

“Alkylsulfonylalkyl” means a radical -(alkylene)-SO₂R where R is alkylas defined above, e.g., methylsulfonylmethyl, ethylsulfonylmethyl, n- oriso-propylsulfonylethyl, and the like.

“Alkylsulfonylamino” means a radical —NHSO₂R where R is alkyl as definedabove, e.g., methylsulfonylamino, ethylsulfonylamino, n- oriso-propylsulfonylamino, and the like.

“Alkylsulfonylaminoalkyl” means a radical -(alkylene)-NHSO₂R where R isalkyl as defined above, e.g., methylsulfonylaminomethyl,ethylsulfonylaminomethyl, n- or iso-propylsulfonylaminoethyl, and thelike.

“Alkoxysulfonylamino” means a radical —NHSO₂R where R is alkoxy asdefined herein, e.g., methoxysulfonylamino, ethoxysulfonylamino, and thelike.

“Alkoxysulfonylaminoalkyl” means a radical -(alkylene)-NHSO₂R where R isalkoxy as defined herein, e.g., methoxysulfonylaminomethyl,ethoxysulfonylaminomethyl, and the like.

“Alkoxy” means a radical —OR where R is alkyl as defined above, e.g.,methoxy, ethoxy, propoxy, or 2-propoxy, n-, iso-, or tert-butoxy, andthe like.

“Alkoxycarbonyl” means a radical —COOR where R is alkyl as definedabove, e.g., methoxycarbonyl, ethoxycarbonyl, and the like.

“Alkoxycarbonylalkyl” means a radical -(alkylene)-COOR where R is alkylas defined above, e.g., methoxycarbonylmethyl, ethoxycarbonylmethyl, andthe like.

“Alkoxyalkyl” means a linear monovalent hydrocarbon radical of one tosix carbon atoms or a branched monovalent hydrocarbon radical of threeto six carbons substituted with at least one alkoxy group, preferablyone or two alkoxy groups, as defined above, e.g., 2-methoxyethyl, 1-,2-, or 3-methoxypropyl, 2-ethoxyethyl, and the like.

“Aminoalkyl” means a linear monovalent hydrocarbon radical of one to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbons substituted with at least one, preferably one or two, —NRR′where R and R′ are independently hydrogen, alkyl, or —COR^(a) whereR^(a) is alkyl, e.g., aminomethyl, methylaminoethyl, 1,3-diaminopropyl,acetylaminopropyl, and the like.

“Aminocarbonylalkyl” means a linear monovalent hydrocarbon radical ofone to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbons substituted with one or two —CONRR′ where R and R′are independently hydrogen, alkyl, or —COR^(a) where R^(a) is alkyl,e.g., aminocarbonylmethyl, methylaminocarbonylmethyl,acetylaminocarbonylpropyl, and the like.

“Alkoxyalkyloxy” means a radical —OR where R is alkoxyalkyl, as definedabove, e.g., 2-methoxyethyloxy, 1-, 2-, or 3-methoxypropyloxy,2-ethoxyethyloxy, and the like.

“Aminoalkyloxy” means a radical —OR where R is aminoalkyl, as definedabove, e.g., 2-aminoethyloxy, 1-, 2-, or 3-methylaminopropyloxy, and thelike.

“Aminocarbonyl” or “carbamoyl” means a radical —CONH₂.

“Aminocarbonylalkyloxy” means a radical —O-(alkylene)-CONRR″ where R andR′ are independently hydrogen or alkyl, as defined above, e.g.,2-aminocarbonylethyloxy, aminocarbonylmethyloxy, and the like.

“Alkylureido” means a radical —NRCONHR′ where R is hydrogen or alkyl andR′ is alkyl, e.g., methylureidomethyl, and the like.

“Alkylureidoalkyl” means a radical -(alkylene)-NRCONHR′ where R ishydrogen or alkyl and R′ is alkyl, e.g., methylureidomethyl, and thelike.

“Aryl” means a monovalent monocyclic or bicyclic aromatic hydrocarbonradical of 6 to 12 ring atoms, and optionally substituted independentlywith one or more substituents, preferably one, two, or threesubstituents, selected from alkyl, haloalkyl, alkoxy, alkylthio, halo,nitro, —COR (where R is alkyl), cyano, amino, alkylamino, dialkylamino,hydroxy, carboxy, or —COOR where R is alkyl. Representative examplesinclude, but are not limited to, phenyl, biphenyl, 1-naphthyl, and2-naphthyl and the derivatives thereof.

“Arylsulfonyl” means a radical —SO₂R where R is aryl as defined above,e.g., phenylsulfonyl, and the like.

“Aralkyl” means a radical -(alkylene)-R where R is an aryl group asdefined above e.g., benzyl, phenylethyl,3-(3-chlorophenyl)-2-methylpentyl, and the like.

“Alkoxycarbamimidoyl” means a radical —C(═NH)NHOR or —C(═NOR)NH₂ where Ris alkyl as defined above, e.g., methoxycarbamimidoyl.

“Cycloalkyl” means a cyclic saturated monovalent hydrocarbon radical ofthree to six carbon atoms, e.g., cyclopropyl, cyclobutyl, and the like,preferably cyclopropyl.

“Carboxyalkyl” means a radical -(alkylene)-COOH, e.g., carboxymethyl,carboxyethyl, 1-, 2-, or 3-carboxypropyl, and the like.

“Carboxyalkyloxy” means a radical —O-(alkylene)-COOH, e.g.,carboxymethyloxy, carboxyethyloxy, and the like.

“Carbamimidoyl” means a radical —C(═NH)NH₂, or a protected derivativethereof.

“Cyanoalkyl” means a radical -(alkylene)-CN, e.g., cyanomethyl,cyanoethyl, cyanopropyl, and the like.

“Dicarboxyalkylaminocarbonylalkyl” means a radical -(alkylene)-CONHRwhere R is alkyl, as defined herein, substituted with two carboxygroups, e.g., —CH₂CONHCH(COOH)(CH₂COOH), —CH₂CONHCH(CH₂COOH)₂,—C(CH₃)₂—CONHCH(COOH)(CH₂COOH), —C(CH₃)₂—CONHCH(CH₂COOH)₂, and the like.

“Dicarboxyalkylamino” means a radical —NHR where R is alkyl, as definedherein, substituted with two carboxy groups

“Dicarboxyalkyaminocarbonylcycloalkyl” means radical of the formula:

where n is 1 to 4 and R is alkyl, as defined herein, substituted withtwo carboxy groups.

“Dialkylamino” means a radical —NRR′ where R and R′ are independentlyalkyl as defined above, e.g., dimethylamino, methylethylamino,methylopropylamino (including all isomeric forms), and the like.

“Dialkylaminosulfonyl” means a radical —SO₂NRR′ where R and R′ areindependently alkyl as defined above, e.g., dimethylaminosulfonyl,methylethylamino-sulfonyl, and the like.

“Dialkylureido” means a radical —NRCONR′R″ where R is hydrogen or alkyland R′ and R″ are independently alkyl, e.g., dimethylureido, and thelike.

“Dialkylureidoalkyl” means a radical -(alkylene)-NRCONR′R″ where R ishydrogen or alkyl and R′ and R″ are independently alkyl, e.g.,dimethylureidomethyl, and the like.

“Guanidinoalkyl” means a linear monovalent hydrocarbon radical of one tosix carbon atoms or a branched monovalent hydrocarbon radical of threeto six carbons substituted with at least one, preferably one or two,—NRC(NRR′)NRR′ where R and R′ are independently hydrogen, alkyl, or—COR^(a) where R^(a) is alkyl, e.g., guanidinomethyl,N′-methylaminoethyl, 2-(N′,N′,N″,N″-tetramethyl-guanidino)-ethyl, andthe like.

“Halo” means fluoro, chloro, bromo, and iodo, preferably fluoro orchloro.

“Haloalkyl” means alkyl substituted with one or more halogen atoms,preferably one to three halogen atoms, preferably fluorine or chlorine,including those substituted with different halogens, e.g., —CH₂Cl, —CF₃,—CHF₂, and the like.

“Haloalkoxy” means a radical —OR where R is haloalkyl as defined above,e.g., —OCH₂Cl, —OCF₃, —OCHF₂, and the like.

“Hydroxyalkyl” means a linear monovalent hydrocarbon radical of one tosix carbon atoms or a branched monovalent hydrocarbon radical of threeto six carbons substituted with one to five hydroxy groups, providedthat if two hydroxy groups are present they are not both on the samecarbon atom. Representative examples include, but are not limited to,hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl,1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl,4-hydroxybutyl, 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2-hydroxyethyl,2,3-dihydroxybutyl, 3,4-dihydroxybutyl and2-(hydroxymethyl)-3-hydroxypropyl, preferably 2-hydroxyethyl,2,3-dihydroxypropyl, and 1-(hydroxymethyl)-2-hydroxyethyl.

“Hydroxyalkyloxy” means a radical —OR where R is hydroxyalkyl as definedabove, e.g., 2-hydroxyethyloxy, 3-hydroxypropyloxy, and the like.

“Hydroxyalkoxyalkylaminocarbonyl” means a radical—CONH-(alkylene)-O-(alkylene)OH where alkylene is as defined above,e.g., —CONH—(CH₂)₂—O—(CH₂)₂OH and the like.

“Heterocycloalkyl” means a saturated or unsaturated monovalent cyclicgroup of 3 to 8 ring atoms in which one or two ring atoms areheteroatoms selected from N, O, or S(O)n, where n is an integer from 0to 2, the remaining ring atoms being C. The heterocycloalkyl ring may beoptionally substituted with one or more substituents, preferably one ortwo substituents, independently selected from alkyl, aryl, heteroaryl,aralkyl, 3,5,6-trihydroxy-2-hydroxymethyl-tetrahydropyran-3-yl,4,5-dihydroxy-2-hydroxymthyl-6-(4,5,6-trihydroxy-2-hydroxymthyl-tetrahydro-pyran-3-yloxy)-tetrahydro-pyran-3-yl,heteroaralkyl, halo, haloalkyl, hydroxy, hydroxyalkyl, alkoxy,alkoxyalkyl, aminoalkyl, guanidinoalkyl, halo, cyano, carboxy, —COOR(where R is alkyl as define above), or —CONR^(a)R^(b) (where R^(a) andR^(b) are independently hydrogen or alkyl), or a protected derivativethereof. More specifically the term heterocycloalkyl includes, but isnot limited to, pyrrolidino, piperidino, morpholino, piperazino,tetrahydropyranyl, and thiomorpholino.

“Heterocycloalkylcarbonyl” means a radical —COR where R isheterocycloalkyl as defined above. More specifically the termheterocycloalkylcarbonyl includes, but is not limited to,1-pyrrolidinocarbonyl, 1-piperidinocarbonyl, 4-morpholinocarbonyl,1-piperazinocarbonyl, 2-tetrahydropyranylcarbonyl, and4-thiomorpholinocarbonyl, and the derivatives thereof.

“Heterocycloalkylcarbonylalkyl” means a radical -(alkylene)-COR where Ris heterocycloalkyl as defined above. More specifically the termheterocycloalkylcarbonyl includes, but is not limited to,1-pyrrolidinocarbonylmethyl, 1-piperidinocarbonylmethyl,4-morpholinocarbonylethyl, 1-piperazinocarbonylmethyl, and thederivatives thereof.

“Heterocycloalkylalkyl” means a radical -(alkylene)-R where R isheterocycloalkyl as defined above. More specifically the termheterocycloalkylalkyl includes, but is not limited to,pyrrolidin-1-ylmethyl, piperidin-1-ylmethyl, 2 morpholin-1-ylethyl,piperazin-1-ylethyl, and the derivatives thereof.

“Heterocycloalkylalkylaminocarbonyl” means a radical —CONH-(alkylene)-Rwhere R is heterocycloalkyl as defined above. More specifically the termheterocycloalkylalkylamino-carbonyl includes, but is not limited to,1-pyrrolidinoethyl-aminocarbonyl, 1-piperidinoethyl-aminocarbonyl,4-morpholinoethylcarbonyl, 1-piperazinoethylaminocarbonyl, and4-thiomorpholinopropylaminocarbonyl, and the derivatives thereof.

“Heteroaryl” means a monovalent monocyclic or bicyclic aromatic radicalof 5 to 10 ring atoms containing one or more, preferably one or two ringheteroatoms selected from N, O, or S, the remaining ring atoms beingcarbon. The heteroaryl ring is optionally substituted with one or moresubstituents, preferably one or two substituents, independently selectedfrom alkyl, haloalkyl, alkoxy, alkylthio, aminoalkyl, guanidinoalkyl,halo, nitro, cyano, amino, alkyl or dialkylamino, hydroxy, carboxy, or—COOR where R is alkyl as define above. More specifically the termheteroaryl includes, but is not limited to, pyridyl, pyrrolyl,imidazolyl, thienyl, furanyl, indolyl, quinolyl, pyrazine, pyrimidine,pyradizine, oxazole, isooxazolyl, benzoxazole, quinoline, isoquinoline,benzopyranyl, and thiazolyl.

“Heteroarylsulfonyl” means a radical —SO₂R where R is heteroaryl asdefined above, e.g., pyridylsulfonyl, furanylsulfonyl, and the like.

“Heteroaralkyl” means a radical -(alkylene)-R where R is a heteroarylgroup as defined above e.g., pyridylmethyl, furanylmethyl,indolylmethyl, pyrimidinylmethyl, and the like.

“Heterocycloamino” means a saturated or unsaturated monovalent cyclicgroup of 3 to 8 ring atoms in which one or two ring atoms areheteroatoms selected from N, O, or S(O)n, where n is an integer from 0to 2, the remaining ring atoms being C provided that at least one of theheteroatom is nitrogen and wherein one or two carbon atoms areoptionally replace by a carbonyl group. The heterocycloamino ring may beoptionally substituted with one or more substituents, preferably one ortwo substituents, independently selected from alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, aminoalkyl, guanidinoalkyl, halo,haloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, haloalkyl, halo,cyano, carboxy, —CONR^(a)R^(b) (where R^(a) and R^(b) are independentlyhydrogen or alkyl), or —COOR where R is alkyl as define above. Morespecifically the term heterocycloamino includes, but is not limited to,pyrrolidino, piperidino, piperazino, and thiomorpholino, and thederivatives thereof.

“Hydroxycarbamimidoyl” means a radical —C(═NH)NHOH or —C(═NOH)NH₂.

The present invention also includes the prodrugs of compounds of FormulaI. The term prodrug is intended to represent covalently bonded carriers,which are capable of releasing the active ingredient of Formula I, whenthe prodrug is administered to a mammalian subject. Release of theactive ingredient occurs in vivo. Prodrugs can be prepared by techniquesknown to one skilled in the art. These techniques generally modifyappropriate functional groups in a given compound. These modifiedfunctional groups however regenerate original functional groups byroutine manipulation or in vivo. Prodrugs of compounds of Formula Iinclude compounds wherein a hydroxy, carbamimidoyl, guanidino, amino,carboxylic, or a similar group is modified. Examples of prodrugsinclude, but are not limited to esters (e.g., acetate, formate, andbenzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) ofhydroxy functional groups in compounds of Formula I and the like.Prodrugs of compounds of Formula I are also within the scope of thisinvention.

The present invention also includes (derivatives and protectedderivatives of compounds of Formula I. For example, when compounds ofFormula I contain an oxidizable nitrogen atom (e.g., when a compound ofFormula I contains a pyridine, amino, alkylamino, piperidino,piperazino, morpholino, or dialkylamino group), the nitrogen atom can beconverted to an N-oxide by methods well known in the art.

Also when compounds of Formula I contain groups such as hydroxy,carboxy, carbonyl, thiol or any group containing a nitrogen atom(s),these groups can be protected with a suitable protecting groups. Acomprehensive list of suitable protective groups can be found in T. W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc.1999, the disclosure of which is incorporated herein by reference in itsentirety. The protected derivatives of compounds of Formula I can beprepared by methods well known in the art.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include:

acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)-benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or

salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. It is understood that thepharmaceutically acceptable salts are non-toxic. Additional informationon suitable pharmaceutically acceptable salts can be found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, which is incorporated herein by reference.

The compounds of the present invention may have asymmetric centers.Compounds of the present invention containing an asymmetricallysubstituted atom may be isolated in optically active or racemic forms.It is well known in the art how to prepare optically active forms, suchas by resolution of materials. Many geometric isomers of olefins, C═Cdouble bonds, and the like can be present in the compounds describedherein, and all such stable isomers are contemplated in the presentinvention. Cis and trans geometric isomers of the compounds of thepresent invention are described and may be isolated as a mixture ofisomers or as separated isomeric forms. All chiral, enantiomeric,diastereomeric, racemic forms and all geometric isomeric forms of astructure (representing a compound of Formula I) are intended, unlessthe specific stereochemistry or isomeric form is specifically indicated.

Certain compounds of Formula I exist in tautomeric equilibrium.Compounds of Formula I, which exist as tautomers are named, illustratedor otherwise described in this application as one possible tautomer.However, it is to be understood that all possible tautomers are meant tobe encompassed by such names, illustrations and descriptions and arewithin the scope of this invention. For example, in compound of FormulaI, the group —C(═NR¹³)NH₂ can tautomerize to —C(═NH)NHR¹³ group.Additionally, as used herein the terms alkyl includes all the possibleisomeric forms of said alkyl group albeit only a few examples are setforth. Furthermore, when the cyclic groups such as aryl, heteroaryl,heterocycloalkyl are substituted, they include all the positionalisomers albeit only a few examples are set forth.

Certain compounds that contain a basic group such as an amine portionand an acid portion e.g., carboxylic acid portion, depending upon the pHof the solution, may exist as a free amine and a free carboxylic acid oras a zwitterion in which the amine is protonated to form an ammonium ionand the carboxylic acid is deprotonated to form a carboxylate ion. Allsuch zwitterions are included in this invention.

“Oxoheterocycloalkyl” means a saturated or unsaturated (provided that itis not aromatic) monovalent cyclic group of 3 to 8 ring atoms in whichone or two ring atoms are heteroatoms selected from N, O, or S(O)n,where n is an integer from 0 to 2, the remaining ring atoms being Cwherein one or two of the carbon atoms is/are replaced with an oxo (C═O)group. The oxoheterocycloalkyl ring may be optionally substituted withone or more substituents, preferably one or two substituents,independently selected from alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, haloalkyl, halo, hydroxy, hydroxyalkyl, alkoxyalkyl,aminoalkyl, guanidinoalkyl, alkoxy, cyano, carboxy, or —COOR where R isalkyl as define above. More specifically the term heterocycloalkylincludes, but is not limited to, 2 or 3-oxopyrrolidin-1-yl, 2, 3, or4-oxopiperidino, 3-oxomorpholino, 2-oxo-piperazino,2-oxotetrahydropyranyl, 3-oxothiomorpholino, 2-imidazolidone, and thederivatives thereof.

“Oxoheterocycloalkylalkyl” means a radical -(alkylene)-R where R is aoxoheterocycloalkylalkyl group as defined above e.g., More specificallythe term oxoheterocycloalkylalkyl; includes, but is not limited to, 2 or3-oxopyrrolidin-1-yl-(methyl, ethyl, or propyl), 2, 3, or4-oxopiperidin-1-yl-(methyl, ethyl, or propyl),3-oxomorpholin4-yl-(methyl, ethyl, or propyl),2-oxopiperazin-1-yl-(methyl, ethyl, or propyl),2-oxotetrahydro-pyran-3-yl-(methyl, ethyl, or propyl),3-oxothiomorpholin-4-yl-(methyl, ethyl, or propyl),2-imidazolidon-1-yl-(methyl, ethyl, or propyl), and the derivativesthereof.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “heterocycloalkyl group optionallymono- or di-substituted with an alkyl group” means that the alkyl maybut need not be present, and the description includes situations wherethe heterocycloalkyl group is mono- or disubstituted with an alkyl groupand situations where the heterocycloalkyl group is not substituted withthe alkyl group.

A “pharmaceutically acceptable carrier or excipient” means a carrier oran excipient that is useful in preparing a pharmaceutical compositionthat is generally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes a carrier or an excipient that is acceptablefor veterinary use as well as human pharmaceutical use. “Apharmaceutically acceptable carrier/excipient” as used in thespecification and claims includes both one and more than one suchexcipient.

“Treating” or “treatment” of a disease includes:

(1) preventing the disease, i.e. causing the clinical symptoms of thedisease not to develop in a mammal that may be exposed to or predisposedto the disease but does not yet experience or display symptoms of thedisease;

(2) inhibiting the disease, i.e., arresting or reducing the developmentof the disease or its clinical symptoms; or

(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

A “therapeutically effective amount” means the amount of a compound ofFormula I that, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

“Thioureido” means a radical —NRC(S)NR′R″ where R, R′, and R″ areindependently hydrogen or alkyl.

“Thioureidoalkyl” means a radical -(alkylene)-NRC(S)NR′R″ where alkyleneis as defined above. Representative examples include but are not limitedto thioureidomethyl, thioureidoethyl, and the like.

“Ureido” means a radical —NHCONH₂.

“Ureidoalkyl” means a radical -(alkylene)-NHCONH₂ where alkylene is asdefined above. Representative examples include but are not limited toureidomethyl, ureidoethyl, and the like.

Representative compounds of this invention where R¹, R² and R^(y) arehydrogen; X¹ is —N—, X², X³, and X⁴ are carbon are disclosed in Table Ibelow.

TABLE I

Posi- Cpd. tion, # R³ R^(x) Position, R^(z) 1

2′-OH 5′- CH₂NHCONH₂ 2

2′-OH 5′-SO₂NH₂ 3

2′-OH 5′-SO₂NH₂ 4

2′-OH 5′-SO₂NH₂

Preferred Embodiments

While the broadest definition of this invention is set forth in theSummary of the Invention, certain compounds of Formula I are preferred.For example:

(I) One preferred group of compounds is represented by the Formula Ia:

wherein R³, R¹³ and R^(z) are as defined in the Summary of theInvention.

Within the above group Ia, a preferred group of compounds is thatwherein R³ is dicarboxyalkylaminocarbonylalkyl, preferably —CH₂CONHR or—C(CH₃)₂CONHR wherein R is dicarboxyalkyl, more preferably1,2-dicarboxyethyl or 1,3-dicarboxyprop-2-yl, even more preferably(R)-1,2-dicarboxyethyl or (S)-1,2-dicarboxyethyl.

Within the above preferred group and more preferred groups containedtherein, an even more preferred group of compounds is that wherein:

R^(z) is halo, hydroxyalkyl, alkylsulfonylamino,alkylsulfonylaminoalkyl, alkylsulfonyl, aminosulfonyl,heterocycloalkylcarbonylalkyl, oxoheterocycloalkyl, carboxyalkyl,oxoheterocycloalkylalkyl, heteroaralkyl, ureido, alkylureido,dialkylureido, ureidoalkyl, alkylureidoalkyl, dialkylureidoalkyl,thioureido, thioureidoalkyl, —COR¹² (where R¹² is alkyl, hydroxyalkyl,or haloalkyl), -(alkylene)-COR¹² (where R¹² is alkyl or haloalkyl),—CONR¹⁴R¹⁵ (where R¹⁴ is hydrogen or alkyl and R¹⁵ is hydrogen, alkyl,aryl, aralkyl, heteroaryl, or heteroaralkyl), -(alkylene)-CONR¹⁶R¹⁷(where R¹⁶ is hydrogen or alkyl and R¹⁷ is hydrogen, alkyl,hydroxyalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl), —NR¹⁸R¹⁹(where R¹⁸ is hydrogen or alkyl and R¹⁹ is hydrogen, alkyl, acyl, aryl,aralkyl, heteroaryl, or heteroaralkyl), -(alkylene)-NR²⁰R²¹ (where R²⁰is hydrogen or alkyl and R²¹ is hydrogen, alkyl, hydroxyalkyl, acyl,aryl, aralkyl, heteroaryl, or heteroaralkyl), —SO₂NR²²R²³ (where R²² andR²³ are independently alkyl), —NR²⁶SO₂NR²⁷R²⁸ (where R²⁶ and R²⁷ areindependently hydrogen or alkyl, and R²⁸ is hydrogen, alkyl, aryl,aralkyl, heteroaryl, or heteroaralkyl or R²⁷ and R²⁸ together with thenitrogen atom to which they are attached form heterocycloamino), or-(alkylene)-NR²⁹SO₂NR³⁰R³¹ (where R²⁹ and R³⁰ are independently hydrogenor alkyl, and R³¹ is hydrogen, alkyl, aryl, aralkyl, heteroaryl, orheteroaralkyl or R³⁰ and R³¹ together with the nitrogen atom to whichthey are attached from heterocycloamino); or

a zwitterion or a pharmaceutically acceptable salt thereof.

Preferably, R^(z) is aminosulfonyl, alkylsulfonylaminoalkyl, halo,carboxyalkyl, hydroxyalkyl, heterocycloalkylcarbonylalkyl, ureido,ureidoalkyl, alkylureidoalkyl, dialkylureidoalkyl, —CONR¹⁴R¹⁵ (where R¹⁴is hydrogen or alkyl and R¹⁵ is hydrogen or alkyl),-(alkylene)-CONR¹⁶R¹⁷ (where R¹⁶ is hydrogen or alkyl and R¹⁷ ishydrogen, alkyl, or hydroxyalkyl), or -(alkylene)-NR²⁰R²¹ (where R²⁰ ishydrogen or alkyl and R²¹ is hydrogen, alkyl, hydroxyalkyl or acyl).

More preferably, R^(z) is fluoro, aminosulfonyl, ureidomethyl,tert-butylureidomethyl, 3,3-dimethylureidomethyl, aminomethyl,piperazin-1-ylcarbonyl-methyl, carboxymethyl,hydroxymethylcarbonylaminomethyl, aminocarbonyl, acetylaminomethyl,aminocarbonylmethyl, methylaminocarbonylmethyl,dimethylaminocarbonylmethyl, 2-hydroxyethylaminocarbonylmethyl,morpholin-4-yl-carbonylmethyl, methoxycarbonylaminomethyl, R or S, orRS-1-hydroxyethylcarbonyl-aminomethyl, hydroxymethyl, ormethylsulfonylaminomethyl.

Within the above preferred group and more preferred, and even morepreferred groups contained therein, a particularly preferred group ofcompounds is that wherein:

R¹³ is hydrogen, hydroxy, methoxy, or ethoxycarbonyl, more preferablyhydrogen.

Most preferably, the compound of Formula I is represented by Formula Ibor Ic:

or a zwitterion or a pharmaceutically acceptable salt thereof.(II) Yet another preferred group of compounds of Formula I are thosewherein the moiety:

is 3′-acetylphenyl, 3′-hydroxyphenyl, 2′-hydroxyphenyl,3′-aminocarbonylphenyl, 3′-cyanophenyl, 5′-fluoro-2′-hydroxyphenyl,5′-chloro-2′-hydroxyphenyl, 2′-hydroxymethylphenyl, 2′-hydroxyphenyl,5′-carboxy-2′-hydroxyphenyl, 2′,5′-dihydroxyphenyl,5′-cyano-2′-methoxyphenyl, 5′-aminocarbonyl-2′-methoxyphenyl,2′,6′-dihydroxyphenyl, 2′-hydroxy-5′-nitrophenyl, 2′-cyanophenyl,3′-hydroxymethylphenyl, 5′-cyano-2′-hydroxy-phenyl,5′-aminocarbonyl-2′-hydroxyphenyl, 2′,6′-dihydroxyphenyl,5′-aminomethyl-2′-hydroxyphenyl, 2′-hydroxy-5′-ureidomethylphenyl,2′-hydroxy-5′-imidazol-2-ylphenyl, 5′-amino-2′-hydroxyphenyl,2′-hydroxy-5′-ureidophenyl,2′-hydroxy-5′-(2-morpholin-4-ylethyl)aminocarbonyl-phenyl,3′-bromo-2′-hydroxy-5′-hydroxymethylphenyl,5′-(2-cyanoethyl)-2′-hydroxyphenyl,3′-bromo-5′-carboxymethyl-2′-hydroxyphenyl,5′-(2-carboxyethyl)-2′-hydroxyphenyl,5′-aminocarbonylmethyl-2′-hydroxyphenyl,3′,5′-dichloro-2′-hydroxyphenyl,2′-hydroxy-5′-[2-(2-hydroxyethoxy)ethylaminocarbonyl]phenyl,5′-dimethylaminosulfonylamino-2′-hydroxy-phenyl,3′-bromo-5′-chloro-2′-hydroxy-phenyl,2′-hydroxy-5′-(4-methylpiperazin-1-ylcarbonyl)phenyl,2′-hydroxy-5′-(4-methylpiperazin-1-ylemthyl)phenyl,5′-carbamimidoyl-2′-hydroxyphenyl,5′-(2-dimethylaminoethylaminocarbonyl)-2′-hydroxyphenyl, or5′-aminocarbonyl-2′-hydroxyphenyl. Preferably 2′-hydroxyphenyl,5′-fluoro-2′-hydroxyphenyl, 5′-chloro-2′-hydroxyphenyl,2′-hydroxymethylphenyl, 2′-hydroxyphenyl, 5′-carboxy-2′-hydroxy-phenyl,2′,5′-dihydroxyphenyl, 2′,6′-dihydroxy-phenyl,2′-hydroxy-5′-nitrophenyl, 5′-cyano-2′-hydroxyphenyl,5′-aminocarbonyl-2′-hydroxyphenyl, 2′,6′-dihydroxyphenyl,5′-aminomethyl-2′-hydroxyphenyl, 2′-hydroxy-5′-ureidomethylphenyl,2′-hydroxy-5′-imidazol-2-ylphenyl, 5′-amino-2′-hydroxyphenyl,2′-hydroxy-5′-ureidophenyl,2′-hydroxy-5′-(2-morpholin-4-ylethyl)aminocarbonyl-phenyl,3′-bromo-2′-hydroxy-5′-hydroxymethylphenyl,5′-(2-cyanoethyl)-2′-hydroxyphenyl,3′-bromo-5′-carboxymethyl-2′-hydroxyphenyl,5′-(2-carboxyethyl)-2′-hydroxyphenyl,5′-aminocarbonylmethyl-2′-hydroxyphenyl,3′,5′-dichloro-2′-hydroxyphenyl,2′-hydroxy-5′-[2-(2-hydroxyethoxy)ethylaminocarbonyl]phenyl,5′-dimethylaminosulfonylamino-2′-hydroxy-phenyl,3′-bromo-5′-chloro-2′-hydroxyphenyl,2′-hydroxy-5′-(4-methylpiperazin-1-ylcarbonyl)phenyl,2′-hydroxy-5′-(4-methylpiperazin-1-ylmethyl)phenyl,5′-carbamimidoyl-2′-hydroxyphenyl,5′-methylaminocarbonylmethyl-2′-hydroxyphenyl,5′-(2-dimethylaminoethylaminocarbonyl)-2′-hydroxyphenyl, or5′-aminocarbonyl-2′-hydroxyphenyl. More preferably,2′,6′-dihydroxyphenyl, 5′-fluoro-2′-hydroxyphenyl,3′-aminosulfonylphenyl, 5′-aminocarbonyl-2′-hydroxyphenyl,5′-aminocarbonylmethyl-2′-hydroxyphenyl,5′-methylaminocarbonylmethyl-2′-hydroxyphenyl,5′-hydroxymethyl-2′-hydroxyphenyl,5′-acetylaminomethyl-2′-hydroxyphenyl, 2′-hydroxy-5′-ureidophenyl;2′-hydroxy-5′-ureidomethylphenyl,2′-hydroxy-5′-N-methylureidomethyl-phenyl,2′-hydroxy-5′-N,N-dimethylureidomethylphenyl, or5′-methylsulfonylamino-2′-hydroxyphenyl.

Within this group, a more preferred group of compounds is that whereinR¹ and R² are hydrogen, X¹ is nitrogen, X²-X⁴ are carbon and R³ isdicarboxyaminocarbonylalkyl, preferably —CH₂CONHR or —C(CH₃)₂CONHRwherein R is 1,2-dicarboxyethyl or 1,3-dicarboxyprop-2-yl, even morepreferably (R)-1,2-dicarboxyethyl.

(III) Yet another preferred group of compounds of Formula I are thosewherein the moiety:

is a group of the formula:

where R^(z) is fluoro, aminosulfonyl, ureidomethyl, —CH₂NHCONCH₃,—CH₂NHCONHC(CH₃)₃, N,N-dimethylureidomethyl, aminomethyl,piperazin-1-ylcarbonylmethyl, carboxymethyl, —CH₂NHCOCH₂OH,aminocarbonyl, acetylaminomethyl, aminocarbonylmethyl,methylaminocarbonylmethyl, dimethylaminocarbonylmethyl,2-hydroxyethylaminocarbonylmethyl, morpholin-4-ylcarbonyl-methyl,methoxycarbonylaminomethyl, hydroxymethyl, or methylsulfonylaminomethyl.

Reference to the preferred embodiments set forth above is meant toinclude all combinations of particular and preferred groups unlessstated otherwise.

General Synthetic Scheme

Compounds of this invention can be made by the methods depicted in thereaction schemes shown below.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCo., (Milwaukee, Wis.), Bachem (Torrance, Calif.), or Sigma (St. Louis,Mo.) or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition) and Larock's ComprehensiveOrganic Transformations (VCH Publishers Inc., 1989). These schemes aremerely illustrative of some methods by which the compounds of thisinvention can be synthesized, and various modifications to these schemescan be made and will be suggested to one skilled in the art havingreferred to this disclosure.

The starting materials and the intermediates of the reaction may beisolated and purified if desired using conventional techniques,including but not limited to filtration, distillation, crystallization,chromatography and the like. Such materials may be characterized usingconventional means, including physical constants and spectral data.

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure over a temperature range from about −78°C. to about 150° C., more preferably from about 0° C. to about 125° C.and most preferably at about room (or ambient) temperature, e.g., about20° C.

Compounds of Formula I in which X¹ is —N—, R¹³ is hydrogen, R³ isdicarboxyalkylaminocarbonylalkyl, and X², X³, X⁴, R¹, R², R^(x), R^(y),and R^(z) are as defined in the Summary of the Invention can be preparedas described in Scheme I below.

Formylation of a phenol derivative of formula 1 (where R is hydrogen orhydroxy protecting group, preferably hydroxy, and R′ is alkyl) providesa compound of formula 2. The formylation reaction is carried out in thepresence of magnesium chloride and an organic base such astriethylamine, and the like, and in a suitable organic solvent such asacetonitrile, and the like. Halogenation of 2 with a suitablehalogenating agent such as N-bromosuccinimide, N-iodosuccinimide, andthe like and in a suitable organic solvent such as dimethylformamide,and the like provides a compound of formula 3 where X is halo.

Compounds of formula 1 are either commercially available or they can beprepared by methods well known in the art. For example, methyl4-hydroxyphenyl-acetate is commercially available.

Protection of the hydroxy group in 3 (where R is hydrogen) with asuitable hydroxy protecting group such as alkyl, methyoxyethoxymethyl,benzyl, and the like, provides a compound of formula 4. A comprehensivelist of other suitable hydroxy protective groups can be found in T. W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, Inc.1999, the disclosure of which is incorporated herein by reference in itsentirety. Preferred hydroxy protecting groups are 2-methoxyethoxymethyland benzyl. The reaction is typically carried out in the presence of abase such as diisopropylethylamine, and the like, and in a halogenatedorganic solvent such as dichloromethane, carbon tetrachloride,chloroform, and the like.

Treatment of 5 with a boronic acid compound of formula 5 where R^(x),R^(y) and R^(z) are as defined in the Summary of the Invention, or aprotected derivative thereof, provides a biphenyl compound of formula 6.The reaction is carried out in the presence of a palladium catalyst suchas tetrakis(triphenylphosphine)palladium and in a suitable organicsolvent such as toluene or dimethoxyethane and a base such as aqueoussodium carbonate, potassium carbonate and the like. Alternatively, thereaction can be carried out in the presence of PdCl₂(dppf).CH₂Cl₂complex in the presence of diisopropylamine in a suitable organicsolvent such as tetrahydrofuran, and the like. Compounds of formula 5are either commercially available or they can be prepared by methodswell known in the art. For example, 5-fluoro-2-methoxyboronic acid iscommercially available. Others can be prepared by treating a halogenatedbenzene of the formula Ph-(R^(x), R^(y) R^(z))X where X is halo andR^(x), R^(y) and R^(z) are as defined in the Summary of the Inventionwith organic metallic reagent such as n-butyl lithium to generate aorganic metallic species which upon treatment with trimethylborateprovides the corresponding boronic acid. Halogenated benzene of theformula Ph-(R^(x), R^(y) R^(z))X is either commercially available or itcan be prepared by methods well known in the art. For example,2-bromo-4-fluorophenol is commercially available.1-(3-Bromo-4-methoxyethoxymethoxybenzyl)-3-tert-butyl urea can beprepared by treating 3-bromo-4-hydroxybenzonitrile withmethoxyethoxymethyl chloride in the presence of a base such asdiisopropylamine, and the like, followed by reduction of the resulting3-bromo-4-methylethoxymethoxy)benzonitrile to3-bromo-4-methoxyethoxymethoxybenzylamine with a suitable reducing agentsuch as diborane. Treatment of 3-bromo-4-methylethoxybenzylamine withtert-butylisocyanate then provides the desired compound.1-(3-Bromo-4-methoxyethoxymethoxybenzyl)-3-tert-butyl urea can beconverted to 1-(3-bromo-4-methoxyethoxymethoxybenzyl)urea by removal ofthe tert-butyl group under acidic hydrolysis reaction conditions.

Condensation of 6 with a 1,2-diamino compound of formula 7 in thepresence of a suitable oxidant such as benzoquinone, air oxidation, orFeCl₃ and O₂ and in a suitable organic solvent such as methanol,ethanol, and the like, provides a compound of formula 8 which uponremoval of the R′ group under basic hydrolysis reaction conditionsprovides a compound of formula 9. Alternatively, the reaction is carriedout utilizing aqueous solution of sodium metabisulfite in an alcoholicsolvent such as isopropanol, and in the presence of oxygen.

Reaction of 9 with dicarboxy-protected dicarboxyalkylamino provides adicarboxy-protected compound of Formula I which upon removal of thedicarboxy protecting groups provides a compound of Formula I where X¹ is—N—. The amination reaction is carried out reacting in the presence of asuitable coupling agent e.g.,benzotriazole-1-yloxytris-pyrrolidinophosphonium hexafluorophosphate(PyBOP®), O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or1,3-dicyclohexylcarbodiimide (DCC), optionally in the presence of1-hydroxybenzotriazole (HOBT), and a base such asN,N-diisopropylethylamine, triethylamine, N-methylmorpholine, and thelike. The reaction is typically carried out at 20 to 30° C., preferablyat about 25° C., and requires 2 to 24 h to complete. Suitable reactionsolvents are inert organic solvents such as N,N-dimethylformamide, andthe like.

Other methods of preparing compounds of Formula (I) are disclosed inU.S. Patent Application Hu, Huiyong et al., Publication No. 20030114457A1 published on Jun. 19, 2003, the disclosure of which is incorporatedherein by reference in its entirety.

Compounds of Formula I in which X¹ is —CH—, R¹³ is hydrogen, R³ isdicarboxyalkylaminocarbonylalkyl, and X², X³, X⁴, R¹, R², R^(x), R^(y),and R^(z) are as defined in the Summary of the Invention can be preparedas described in Scheme II below.

Protection of the hydroxy group in a compound of formula 7 with asuitable hydroxy protecting group provides a compound of formula 11. Acomprehensive list of suitable hydroxy protective groups can be found inT. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons,Inc. 1999, the disclosure of which is incorporated herein by referencein its entirety. Preferred hydroxy protecting group is2-methoxyethoxymethyl. The reaction is typically carried out in thepresence of a base such as N,N-diisopropylethylamine, and the like, andin a halogenated organic solvent such as dichloromethane, carbontetrachloride, chloroform, and the like.

Ethynylation of 11 utilizing a modified procedure described in Muller,S.; Liepold, B.; Roth G. J.; Bestmann H. J. Synlett 1996, 6, 521-522provides a ethynylbiphenyl compound of formula 12.

Reaction of a compound of formula 12 with a cyano compound of formula 13where PG¹ is a suitable nitrogen protecting group such asmethylsulfonyl, tert-butoxycarbonyl, trifluoroacetyl, and the like, andX is halo, utilizing the reaction conditions described in Sakamoto, T;Kondo, Y.; Iwashita, S.; Nagano, T.; Yamanaka, H. Chem. Pharm. Bull.1988, 36, 1305 provides 5-cyano-2-biphenyl-3-ylindole compound offormula 14 (where X¹, X², X³ and X⁴ are carbon and PG¹ is not hydrogen).Deprotection of the amino group in 14 provides a5-cyano-2-(biphenyl-3-yl)-1H-indole compound of formula 15. The reactionconditions utilized in the deprotection step depends on the nature ofthe nitrogen protecting group. For example, if the protecting group ismethylsulfonyl it is removed under basic hydrolysis reaction conditions.Suitable bases are aqueous sodium hydroxide, potassium hydroxide, andthe like. The reaction is carried out in an alcoholic solution such asmethanol, ethanol, and the like. If the protecting group istert-butoxycarbonyl it is removed under acidic hydrolysis reactionconditions. Compounds of formula 13 are either commercially available orthey can be prepared by methods well known in the art.

The hydroxy-protecting group in 15 is then removed to provide5-cyano-2-(2-hydroxybiphenyl-3-yl)-1H-indole 16. The reaction conditionsemployed for the deprotection reaction depend on the nature of thehydroxy protecting group. For example, if the protecting group is2-methoxyethoxymethyl, it is removed by treating 16 with an acid undernon-aqueous reaction conditions, in a suitable alcoholic solvent.

The cyano group in compound 16 is then converted into the carbamimidoylgroup by first treating 16 with hydrogen chloride gas in an anhydrousalcoholic solvent such as methanol, ethanol and the like, and thentreating the resulting(5-methoxycarbonimidolyl)-2-(2-hydroxybiphenyl-3-yl)-1H-indole 17 withan inorganic base such as ammonium carbonate, and the like in analcoholic solvent such as methanol, ethanol, or with excess ammonia togive resulting (5-carbamimidolyl)-2-(2-hydroxybiphenyl-3-yl)-1H-indoleof formula 18 which is then converted to a compound of Formula I asdescribed in Scheme I above.

Compounds of Formula I can be converted to other compounds of Formula I.For example, a compound of Formula I where R^(x) is alkoxy, can beconverted to corresponding compound of Formula I where R^(x) is hydroxyby hydrolysis of the alkoxy group by a suitable dealkylating reagentsuch as hydrobromic acid, and the like. A compound of Formula I whereR^(z) is cyano can be converted to a corresponding compound of Formula Iwhere R^(z) is aminocarbonyl under hydrolysis reaction conditions. Thecyano group can also be reduced to give aminomethyl group which can betreated with isocyanate or thiocyanate to give corresponding compound ofFormula I where R^(z) is ureidomethyl or thioureidomethyl respectively.A compound of Formula I where R¹³ is hydrogen can be converted to acorresponding compound of Formula I where R¹³ is hydroxy or alkoxy byreacting it with hydroxylamine or alkoxyamine under conditions wellknown in the art.

Utility

The compounds of this invention inhibit Factors VIIa, IXa, Xa, and XIa,in particular Factor VIIa, and are therefore useful as anticoagulantsfor the treatment or prevention of thromboembolic disorders in mammals.

Particular disease states which may be mentioned include the therapeuticand/or prophylactic treatment of venous thrombosis (e.g. DVT) andpulmonary embolism, arterial thrombosis (e.g. in myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis), and systemic embolism usually from the atrium during atrialfibrillation or from the left ventricle after transmural myocardialinfarction, or caused by congestive heart failure; prophylaxis ofreocclusion (i.e., thrombosis) after thrombolysis, percutaneoustrans-luminal angioplasty (PTA) and coronary bypass operations; theprevention of rethrombosis after microsurgery and vascular surgery ingeneral.

Further indications include the therapeutic and/or prophylactictreatment of disseminated intravascular coagulation caused by bacteria,multiple trauma, intoxication or any other mechanism; anticoagulanttreatment when blood is in contact with foreign surfaces in the bodysuch as vascular grafts, vascular stents, vascular catheters, mechanicaland biological prosthetic valves or any other medical device; andanticoagulant treatment when blood is in contact with medical devicesoutside the body such as during cardiovascular surgery using aheart-lung machine or in haemodialysis; the therapeutic and/orprophylactic treatment of idiopathic and adult respiratory distresssyndrome, pulmonary fibrosis following treatment with radiation orchemotherapy, septic shock, septicemia, inflammatory responses, whichinclude, but are not limited to, edema, acute or chronic atherosclerosissuch as coronary arterial disease and the formation of atheroscleroticplaques, cerebral arterial disease, cerebral infarction, cerebralthrombosis, cerebral embolism, peripheral arterial disease, ischaemia,angina (including unstable angina), reperfusion damage, restenosis afterpercutaneous trans-luminal angioplasty (PTA) and coronary artery bypasssurgery.

The compounds of Formula I can also be used in the treatment of canceror rheumatoid arthritis.

Testing

The ability of the compounds of this invention to inhibit factor VIIaand Xa can be tested in vitro and in vivo assays described in biologicalassays Example 1 and 2 below.

Administration and Pharmaceutical Compositions

In general, the compounds of this invention will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the compound of this invention, i.e., the active ingredient,will depend upon numerous factors such as the severity of the disease tobe treated, the age and relative health of the subject, the potency ofthe compound used, the route and form of administration, and otherfactors.

Therapeutically effective amounts of compounds of Formula I may rangefrom approximately 0.01-50 mg per kilogram body weight of the recipientper day; preferably about 0.1-20 mg/kg/day, even more preferably about0.25 mg/kg/day to 10 mg/kg/day. Thus, for administration to a 70 kgperson, the dosage range would most preferably be about 7 mg to 1.4 gper day.

In general, compounds of this invention will be administered aspharmaceutical compositions by any one of the following routes: oral,systemic (e.g., transdermal, intranasal or by suppository), orparenteral (e.g., intramuscular, intravenous or subcutaneous)administration. The preferred manner of administration is oral orparenteral using a convenient daily dosage regimen, which can beadjusted according to the degree of affliction. Oral compositions cantake the form of tablets, pills, capsules, semisolids, powders,sustained release formulations, solutions, suspensions, elixirs,aerosols, or any other appropriate compositions.

The choice of formulation depends on various factors such as the mode ofdrug administration (e.g., for oral administration, formulations in theform of tablets, pills or capsules are preferred) and thebioavailability of the drug substance. Recently, pharmaceuticalformulations have been developed especially for drugs that show poorbioavailability based upon the principle that bioavailability can beincreased by increasing the surface area i.e., decreasing particle size.For example, U.S. Pat. No. 4,107,288 describes a pharmaceuticalformulation having particles in the size range from 10 to 1,000 nm inwhich the active material is supported on a crosslinked matrix ofmacromolecules. U.S. Pat. No. 5,145,684 describes the production of apharmaceutical formulation in which the drug substance is pulverized tonanoparticles (average particle size of 400 nm) in the presence of asurface modifier and then dispersed in a liquid medium to give apharmaceutical formulation that exhibits remarkably highbioavailability.

The compositions are comprised of in general, a compound of Formula I incombination with at least one pharmaceutically acceptable excipient.Acceptable excipients are non-toxic, aid administration, and do notadversely affect the therapeutic benefit of the compound of Formula I.Such excipient may be any solid, liquid, semi-solid or, in the case ofan aerosol composition, gaseous excipient that is generally available toone skilled in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols.

Compressed gases may be used to disperse a compound of this invention inaerosol form. Inert gases suitable for this purpose are nitrogen, carbondioxide, etc.

Other suitable pharmaceutical excipients and their formulations aredescribed in Remington's Pharmaceutical Sciences, edited by E. W. Martin(Mack Publishing Company, 18th ed., 1990).

The amount of the compound in a formulation can vary within the fullrange employed by those skilled in the art. Typically, the formulationwill contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt% of a compound of Formula I based on the total formulation, with thebalance being one or more suitable pharmaceutical excipients.Preferably, the compound is present at a level of about 1-80 wt %.Representative pharmaceutical formulations containing a compound ofFormula I are described below.

The compounds of Formula I can be administered alone or in combinationwith other compounds of Formula I or in combination with one or moreother active ingredient(s). For example, a compound of Formula I can beadministered in combination with another anticoagulant agent(s)independently selected from a group consisting of a thrombin inhibitor,a factor IXa, and a factor Xa inhibitor. Preferably, the thrombininhibitor is Inogatran®, Melagatran® or prodrugs thereof which aredisclosed in PCT Application Publication Nos. WO 94/29336 and WO97/23499, the disclosures of which are incorporated herein by referencein their entirety. Factor Xa inhibitors that may be used in thecombination products according to the invention include those describedin Current Opinion in Therapeutic Patents, 1993, 1173-1179 and ininternational patent applications WO 00/20416, WO 00/12479, WO 00/09480,WO 00/08005, WO 99/64392, WO 99/62904, WO 99/57096, WO 99/52895, WO99/50263, WO 99/50257, WO 99/50255, WO 99/50254, WO 99/48870, WO99/47503, WO 99/42462, WO 99/42439, WO 99/40075, WO 99/37304, WO99/36428, WO 99/33805, WO 99/33800, WO 99/32477, WO 99/32454, WO99/31092, WID 99/26941, WO 99/26933, WO 99/26932, WO 99/26919, WO99/26918, WO 99/25720, WO 99/16751, WO 99/16747, WO 99/12935, WO99/12903, WO 99/11658, WO 99/11617, WO 99/10316, WO 99/07732, WO9/07731, WO 99/05124, WO 99/00356, WO 99/00128, WO 99/00127, WO99/00126, WO 9/00121, WO 98/57951, WO 98/57937, WO 98/57934, WO98/54164, WO 98/46591, WO 98/31661, WO 98/28282, WO 98/28269, WO98/25611, WO 98/24784, WO 98/22483, WO 98/16547, WO 98/16525, WO98/16524, WO 98/16523, WO 98/15547, WO 98/11094, WO 98/07725, WO98/06694, WO 98/01428, WO 7/48706, WO 97/46576, WO 97/46523, WO97/38984, WO 97/30971, WO 97/30073, WO 97/29067, WO 97/24118, WO97/23212, WO 97/21437, WO 97/08165, WO 97/05161, WO 96/40744, WO96/40743, WO 96/40679, WO 96/40100, WO 96/38421, WO 96/28427, WO96/19493, WO 96/16940, WO 95/28420, WO 94/13693, WO 00/24718, WO99/55355, WO 99/51571, WO 99/40072, WO 99/26926, WO 98/51684, WO97/48706, WO 97/24135, WO 97/11693, WO 00/01704, WO 00/71493, WO00/71507, WO 00/71508, WO 00/71509, WO 00/71511, WO 00/71512, WO00/71515, WO 00/71516, WO 00/13707, WO 00/31068, WO 00/32590, WO00/33844, WO 00/35859, WO 00/35886, WO 00/38683, WO 00/39087, WO00/39092, WO 00/39102, WO 00/39108, WO 00/39111, WO 00/39117, WO00/39118, WO 00/39131, WO 00/40548, WO 00/40571, WO 00/40583, WO00/40601, WO 00/47207, WO 00/47553, WO 00/47554, WO 00/47563, WO00/47578, WO 00/51989, WO 00/53264, WO 00/59876, WO 00/59902, WO00/71510, WO 00/76970, WO 00/76971, WO 00/78747, WO 01/02356, WO01/02397, WO 01/05784, WO 01/09093, WO 01/12600, WO 01/19788, WO01/19795, WO 01/19798, WO 93/15756, WO 94/17817, WO 95/29189, WO96/18644, WO 96/20689, WO 96/39380, WO 97/22712, WO 97/36580, WO97/36865, WO 97/48687, WO 98/09987, WO 98/46626, WO 98/46627, WO98/46628, WO 98/54132, WO 99/07730, WO 99/33458, WO 99/37643 and WO99/64446; in U.S. Pat. Nos. 6,034,093, 6,020,357, 5,994,375, 5,886,191,5,849,519, 5,783,421, 5,731,315, 5,721,214, 5,693,641, 5,633,381,5,612,378, 6,034,127, 5,670,479, 5,658,939, 5,658,930, 5,656,645,5,656,600, 5,639,739, 5,741,819, 6,057,342, 6,060,491, 6,080,767,6,087,487, 6,140,351, 6,395,731, and 5,646,165; in Japanese patentapplications Nos. JP 99152269, JP 10017549, JP 10001467, JP 98017549, JP00178243, JP 11140040, JP 12143623, JP 12204081, JP 12302765, JP 6327488and JP 98001467; in European patent applications EP 937 723, EP 937 711,EP 874 629, EP 842 941, EP 728 758, EP 540 051, EP 419 099, EP 686 642,EP 1 016 663 and EP 529 715; and in German patent applications Nos. DE19845153, DE 19835950, DE 19743435, DE 19829964, DE 19834751, DE19839499, DE19900355, DE19900471 and DE 19530996, the specific andgeneric disclosures in all of which documents are hereby incorporated byreference.

Factor Xa inhibitors also include those disclosed in internationalpatent applications WO 96/10022, WO 97/28129, WO 97/29104, WO 98/21188,WO 99/06371, WO 99/57099, WO 99/57112, WO 00/47573, WO 00/78749, WO99/09027 and WO 99/57113, the specific and generic disclosures in all ofwhich documents are hereby incorporated by reference, as well as4-{4-[4-(5-chloroindol-2-ylsulfonyl)piperazine-1-carbonyl]phenyl}-pyridine-1-oxideand pharmaceutically acceptable derivatives thereof. Preferred Factor Xainhibitors include antistatin, tick anticoagulant protein and thoseknown as SQ-311 and SQ-315 (see international patent application WO98/57951); SN-292 (see international patent application WO 98/28282);SN-429 and SN 116 (see international patent application WO 98/28269);RPR-208707 (see international patent application WO 98/25611 at Example48); XU-817 (see international patent application WO 98/01428); SF-324and SF-303 (see international patent application WO 97/23212); YM 60828(see international patent application WO 96/16940 at Example 75);FACTOREX (see U.S. Pat. No. 5,783,421); SF-324 (see European patentapplication EP 874 629); DX9065A (see European patent application EP 540051 at Example 39);1-(4-carbamimidoylbenzyl)-4-(6-chloronaphthalene-2-ylsulfonyl)-piperazin-2-one(see JP 12204081 at Example 2); M55555 (see international patentapplication WO 99/33805 at Example 39); DPC423(1-(3-carbamimidoylphenyl)-2-(2′-aminolsulfonyl[1,1′-biphenyl]-4-ylaminocarbonyl)-4-bromopyrrole,see international patent application WO 98/28269);3-(3,5-difluoro-6-[3-(4,5dihydro-1-methyl-imidazol-2-yl)-phenoxy]-4-[2,3-dihydroxy-propoxy]-pyridin-2-yloxy)-4-hydroxy-benzamidine(see international patent application WO 00/31068); ZK-807834 (seeinternational patent application WO 7/29067);1,4-diaza-4-(6-chloronaphthalene-2-ylsulfonyl)-6-(methoxymethyl)-7-oxa-1′-(pyridin-4-yl)spiro[bicyclo-[4-3.0]-nonane-8,4′-piperidine]-2-one(see international patent application WO 01/02397);(S)-1-(4-aminoquinazolin-7-ylmethyl)-4-[2-(5-chlorothien-2-yloxy)acetyl]-3-methoxy-methylpiperazin-2-one(see international patent application WO 00/32590);3-(2-[4-(2-aminosulfonyl-phenyl)benzoylphenoxy)-benzamidine (seeinternational patent application WO 01/19788); and4-(2-[4-(5-chloroindol-2-yl-sulfonyl)-2-(pyrrolidin-1-ylcarbonylmethyl)piperazin-1-yl-carbonyl]-thiazol-5-yl)pyridineN-oxide (see Japanese patent application No. JP 12143623); as well asthe compounds of Example 7 of international patent application WO98/21188, of Examples 3 and 6 of WO 99/57113, of Example 6 ofinternational patent application WO 00/78747, of Examples 188, 211 and167 of U.S. Pat. No. 6,080,767, of Examples 40, 54 and 55 ofinternational patent application WO 99/33805, of Examples 5, 6, 8, 9,10, 11, 12, 13, 15, 16 and 17 of international patent application WO01/05784, of Examples 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 22,23, 25, 26, 28, 29, 30, 31, 32, 33, 34, 38, 39, 40, 41, 42 and 43 ofinternational patent application WO 01/12600, and of Examples 802 and877 of international patent application WO 00/35886. Other anticoagulantagents that can be used in the combination therapy are those disclosedin U.S. Patent Applications Publication Nos. 20020065303, 20020061842,20020058677, 20020058657, 20020055522, 20020055469, 20020052368,20020040144, 20020035109, 20020032223, 20020028820, 20020025963,20020019395, 20020019394, 20020016326, 20020013314, 20020002183,20010046974, 20010044537, 20010044536, 20010025108, 20010023292,20010023291, 20010021775, 20010020020033, 20010018423, 20010018414, and20010000179, which are incorporated herein by reference in theirentirety.

Suitable formulations for use in administering melagatran andderivatives (including prodrugs) thereof are described in theliterature, for example as described in inter alia international patentapplications WO 94/29336, WO 96/14084, WO 96/16671, WO 97/23499, WO97/39770, WO 97/45138, WO 98/16252, WO 99/27912, WO 99/27913, WO00/12043 and WO 00/13671, the disclosures in which documents are herebyincorporated by reference.

Similarly, suitable formulations for use in administering Factor Xainhibitors and derivatives (including prodrugs) thereof are described inthe literature, for example as described in the prior art documentsrelating to Factor Xa inhibitors that are mentioned hereinbefore, thedisclosures in which documents are hereby incorporated by reference.Otherwise, the preparation of suitable formulations, and in particularcombined preparations including both melagatran/derivative and Factor Xainhibitor/derivative may be achieved non-inventively by the skilledperson using routine techniques. The amounts of melagatran, Factor Xainhibitor, or derivative of either, in the respective formulation(s)will depend on the severity of the condition, and on the patient to betreated, as well as the compound(s) which is/are employed, but may bedetermined non-inventively by the skilled person.

Suitable doses of melagatran, Factor Xa inhibitors and derivatives ofeither, in the therapeutic and/or prophylactic treatment of mammalian,especially human, patients may be determined routinely by the medicalpractitioner or other skilled person, and include the respective dosesdiscussed in the prior art documents relating to melagatran (orderivatives (including prodrugs) thereof), and to Factor Xa inhibitors,that are mentioned hereinbefore, the disclosures in which documents arehereby incorporated by reference.

EXAMPLES

All solvents and reagents were purchased from Aldrich and used asreceived except where noted. All reactions and products were analyzedusing HPLC, employing an Agilent HP1100 system fitted with a diode arraydetector and a Phenomenex Prodigy 5μ ODS-3 100A column, 150 mm×3.0 mm ID[Phenomenex catalogue #00D4096-Y0]. Chromatographic runs were performedat column temperatures of 40° C. and compound detection was performed atboth 214 and 254 nm. Gradient elution was employed, usingacetonitrile-water mobile phase systems with TFA as acid buffer,typically over 5-10 minute gradients. Moisture content of reactionproducts and reagents were determined using an EM-Science model V-200AQUASTAR volumetric Karl Fischer titrator. Combustion analyses wereperformed by Robertson Microlit Laboratories, Inc., Madison, N.J.

Reference A Synthesis of methyl(3-bromo-5-formyl-4-hydroxyphenyl)acetate

Method A Step 1

To a 12 L round bottom flask with overhead stirrer, condenser,thermocouple, and heating mantle was added methyl 4-hydroxyphenylacetate(302 g, 1.82 mol) and acetonitrile (3 L). The reaction mixture wasstirred until the solids had dissolved, then triethylamine (1015 mL, 737g, 7.28 mol) was added. Anhydrous magnesium chloride (346 g, 3.63 mol)was then added in portions over a 5-minute period, resulting in anincrease in the internal temperature to ˜55° C. The reaction mixture washeated to reflux (internal temperature: 68° C.) for 1 h, resulting in abrownish solution with some suspended white solids. Paraformaldehyde(382 g, 12.7 mol, Aldrich catalogue number 44,124-4) was added and theheating was continued for 16-24 h until HPLC-UV analysis shows >95%consumption of methyl 4-hydroxyphenylacetate. The reaction mixture wasdiluted with diethyl ether (3 L), and 1N aqueous HCl (3 L) was added,resulting in dissolution of the magnesium salts. The layers wereseparated and the organic phase was washed with 1N aqueous HCl, followedby saturated aqueous sodium chloride. The organic was dried (Na₂SO₄) andconcentrated to give (3-formyl-4-hydroxyphenyl)acetic acid methyl ester(308 g; 87.3%) as an amber oil.

Step 2

To a 2 L round bottom flask with magnetic stirrer, thermocouple, and icewater bath was added (3-formyl-4-hydroxyphenyl)acetic acid methyl ester(308 g, 1.59 mol) and N,N-dimethylformamide (1 L). The reaction mixturewas stirred and cooled to −5 to 5° C. A solution of N-bromosuccinimide(311 g, 1.75 mol) in DMF (1.5 to 2 L) was added dropwise, at such a rateto keep the internal temperature between 5 and 10° C. Once the additionwas complete, the reaction mixture was allowed to warm to roomtemperature and monitored for completion by HPLC analysis. The reactiontime was about 4 h. The reaction mixture was diluted withisopropylacetate (3 L), and the resulting solution was extracted withwater to remove the DMF. Concentration of the organic phase afforded areddish solid that was dissolved in hot isopropanol (400 mL) and allowedto cool while stirring. The resulting off-white crystalline product wasfiltered and washed with cold IPA (−20° C.; 400 mL) to yield methyl(3-bromo-5-formyl-4-hydroxyphenyl)acetate (368 g, 84.7%) as off-whitecrystals.

Method B: Alternative method for the synthesis of methyl(3-bromo-5-formyl-4-hydroxyphenyl)-acetate Step 1

A 3-neck, 3 L round-bottom flask equipped with an over-head stirrer,thermometer, reflux condensor, and N₂ line was charged with(4-hydroxyphenyl)-acetic acid methyl ester (Aldrich; 166.2 g, 1.00 mol)and acetonitrile (1000 mL). Triethylamine (121.4 g, 1.20 mols, 1.2 eq.)was added in a single portion to the stirring solution and thenmagnesium chloride (98% w/w; 115.2 g, 1.10 mols, 1.1 eq.) was addedportionwise over 15 min. The reaction mixture was stirred for 20 min.and then heated to 75° C. for 1 h during which the magnesium saltsdissolved. Paraformaldehyde (95% w/w as prills; Aldrich #44,124-4; 63.2g, 2.00 mols, 2.00 eq.) was added portionwise over 30 min. After heatingto 80° C. for 2 h, the reaction was found to be incomplete and hence afurther portion of paraformaldehye (6.00 g, 0.20 mols, 0.2 eq.) wasadded portion-wise. After 2 h, the reaction mixture was concentrated insitu (T_(int)≦70° C.; approx. 30-40 Torr) to approx. ⅓ its originalvolume and then allowed to cool to room temperature. Water (500 mL) wasadded over a few minutes and a thick yellow slurry formed. Uponreturning to ˜30° C. conc. phosphoric acid (85% w/w; 230.7 g, 2.00 mols)was added portion-wise and the mixture was stirred vigorously. Most ofthe solids dissolved and after cooling (T_(int)˜20° C.) isopropylacetate (500 mL) was added in a single portion. The mixture was stirredvigorously until all the solids dissolved and the layers separatedcleanly. The solution was transferred to a separating funnel and theaqueous layer was separated. The organic layer was washed with a mixtureof water and conc. phosphoric acid (115 g, 1.00 mol) and the organiclayer containing a mixture of methyl 3-formyl-4-hydroxyphenylacetate andmethyl 4-hydroxyphenylacetate was separated and used in the next step.

Step 2

The solution containing a mixture of methyl3-formyl-4-hydroxyphenylacetate and methyl 4-hydroxyphenylacetate (˜900mL total volume, containing 500 mL ispropyl acetate with ˜300 mLacetonitrile) was transferred back to the original reaction vessel andacetonitrile (900 mL) added. The reaction was stirred and heated to 60°C. and a mixture of sodium hydrogensulfite (NaHSO₃; Aldrich #24, 397-3,58.5% SO₂; 109.4 g, 1.00 mols) dissolved in water (175 mL) was added ina single portion to the stirring reaction and heating was continued.Within 5 min, a white precipitate formed. After 16 h, the reactionmixture was allowed to cool gradually to T_(int): ˜5° C. Theprecipitates were collected and the filter cake washed with cold 1:1acetonitrile-isopropyl acetate and dried to give sodium salt of4-hydroxy-3-(hydroxysulfomethyl)phenyl]-acetic acid methyl ester (208 g,70%).

Step 3

Sodium salt of 4-hydroxy-3-(hydroxysulfomethyl)phenyl]-acetic acidmethyl ester (205.2 g, 0.688 mol) was dissolved in water (2 L) at roomtemperature and stirred. Conc. phosphoric acid (400 mL) was added in asingle portion and the mixture turned cloudy within minutes. Thereaction mixture was extracted with dichloromethane and the combinedextracts were concentrated to yield methyl(3-formyl-4-hydroxyphenyl)acetate (112 g, 58%) which was converted tothe title compound as described in Method A, Step 2 above.

Reference B Synthesis of 3,4-diaminobenzamidine monohydrochloride

Step 1

A mixture of 4-amino-3-nitrobenzonitrile (63.3 g, 388 mmol) in1,4-dioxane (600 mL) and anhydrous ethanol (600 mL) was cooled in an icewater bath to 0-5° C. and treated with gaseous HCl for 1.5 h. Thereaction mixture was tightly sealed and allowed to warm up to roomtemperature with stirring for 18 h. The flask was then carefullyunsealed and the reaction mixture was diluted with anhydrous diethylether (about 2.4 L) until a cloudy solution was obtained. A minimumamount of absolute ethanol required to give a clear solution was thenadded, and the resulting solution stirred until crystals of4-amino-3-nitro-benzimidic acid ethyl ester were observed. Ether wasthen cautiously added to complete the crystallization process and thesuspension was allowed to stand for about 30 minutes. The crystals werefiltered and washed with dry diethyl ether, then allowed to dry underaspirator vacuum. The crystals were dried in vacuo to give4-amino-3-nitro-benzimidic acid ethyl ester hydrochloride (84.6 g) asoff-white crystals.

Step 2

4-Amino-3-nitro-benzimidic acid ethyl ester hydrochloride (84.5 g, 344mmol) was suspended in absolute ethanol (750 mL) and then cooled to 0°C. Ammonia was then passed through the solution for a period of 2 h. Theflask was tightly sealed and allowed to warm up to room temperature overan 18 h period with stirring. The product was crystallized with diethylether and the resulting solid was filtered, washed and dried to give4-amino-3-nitrobenzamidine monohydrochloride (70.7 g) as an off-whitepowder.

Step 3

A suspension of 4-amino-3-nitrobenzamidine monohydrochloride (15 g, 69mmol) and Pearlman's catalyst [Pd(OH)₂, 1.0 g, 7.12 mmol) in methanol(200 mL) was shaken under hydrogen atmosphere 50 psi for 1.5 h. Thesuspension was filtered through Celite and the filtrate was addeddropwise to anhydrous diethyl ether (400 mL) to precipitate3,4-diaminobenzamidine monohydrochloride as a tan solid.

Reference C Synthesis of N-tert-butyl4-methoxy-5-(benzenesulfonamido)-3-boronic acid

Step 1

A solution of 2-iodoanisole (221.2 g, 966 mmol) in dichloromethane (2.3L) was cooled to 0° C. and chlorosulfonic acid (64.5 mL, 112.6 g, 966mmol) was added dropwise with stirring over a 15-minute period. Thereaction mixture was allowed to warm to 10° C. over 3 h. Nitrogen gaswas passed over the solution and the outlet was bubbled through asolution of aqueous sodium hydroxide to scrub the gaseous hydrogenchloride produced in the reaction. An aliquot of the reaction wasanalyzed by HPLC, which showed that 2-iodoanisole had been consumed. Thereaction mixture was treated with phosphorus pentachloride (217.8 g,1.045 mol) and stirred at room temperature for 2 h. The reaction mixturewas concentrated in vacuo to remove most of the volatile components thenfurther concentrated at a bath temperature of 100° C. to remove POCl₃produced in the reaction. The resulting oily residue was dissolved inCH₂Cl₂ (2.8 L) and this solution was stirred with water (3 L) whilesolid sodium bicarbonate was added to maintain the pH around 7. Thelayers were separated and the organic phase was cooled to 0° C., thentert-butylamine (230 mL, 160 g) was added at such a rate to maintain theinternal temperature≦10° C. The reaction mixture was allowed to warm upto ambient temperature overnight, then washed with 5% sodium hydroxide.The organic phase was concentrated in vacuo to give N-tert-butyl3-iodo-4-methoxybenzenesulfonamide (340 g) as an off-white solid.

Step 2

N-tert-Butyl 3-iodo-4-methoxybenzenesulfonamide (335 g, 907 mmol) wasdissolved in dichloromethane (3 L) and the resulting solution was cooledto an internal temperature of −20° C. The solution was treated with a3.0 M solution of methylmagnesium bromide in diethyl ether (308 mL, 925mmol) dropwise over 0.5 h to maintain the internal temperature of theflask at −20±5° C. The reaction mixture was allowed to stir at −20±5° C.for 2.5 h then a 2.13 M solution of isopropylmagnesium bromide indiethyl ether (511 mL, 1.09 mol) was added at about −35° C. Theresulting solution was allowed to stir at −35±5° C. for 1.5 h. Thereaction mixture was warmed to 0° C. and additional isopropylmagnesiumbromide in diethyl ether (86.0 mL, 183 mmol) was added. The reactionmixture was stirred for 2 h at 0° C., then an additional aliquot ofisopropylmagnesium bromide in diethyl ether (25.0 mL; 53.3 mmol) wasadded. The reaction mixture was treated with trimethylborate (320 mL;2.90 mol) in THF (175 mL) in one portion, resulting in a temperatureincrease to 27° C. The reaction mixture was stirred at this temperaturefor 4 h, then poured into water (1.3 L) and 85% phosphoric acid wasadded until the solution was pH 2. The layers were separated and theorganic phase was washed with 1.5 N aqueous NaOH (2 L), followed by 1%aqueous NaOH (2 L). The combined aqueous phases were acidified withphosphoric acid to pH 2 and the resulting acidic solution was extractedwith 9:1 dichloromethane/THF solution (2 L followed by 1 L). The organicphase was dried (Na₂SO₄), filtered and concentrated in vacuo to giveabout 250 g of a white solid which was dissolved in ethanol (1 L). Thesolution was diluted with water to give a total volume of 4 L and theresulting solution was stirred at room temperature overnight. Theresulting crystalline solid was filtered and dried under high vacuumovernight to afford N-tert-butyl4-methoxy-5-(benzenesulfonamido)-3-boronic acid (221 g) as a whitesolid, which was a dihydrate (approximately). The filtrate was extractedwith a 9:1 solution of dichloromethane/THF and the extract evaporated.The crude solid (23 g) was recrystallized from a 3:1 solution ofwater/ethanol (500 mL) to yield an additional 19 g of product as a whitesolid.

Reference D Synthesis of methyl(5′-N-tert-butylsulfamoyl-5-formyl-2′-methoxy-6-methoxyethoxymethoxybiphenyl-3-yl)acetate

Step 1

To a 5-L round-bottom flask was added methyl(3-bromo-5-formyl-4-hydroxy-phenyl)acetate (210 g, 769 mmol, prepared inReference A above), dichloromethane (2 L), and N,N-diisopropylethylamine(161 mL, 119 g, 923 mmol). A solution of 2-methoxyethoxymethyl chloride(MEM chloride, obtained from TCI America; 106 mL; 923 mmol) indichloromethane (500 mL) was added dropwise via an addition funnel over2 h. The reaction was allowed to stir at ambient temperature forovernight, after which time HPLC analysis showed that the reaction wascomplete. The reaction mixture was diluted with 0.5 N aqueous HCl (1 L)and the solution was allowed to stir at ambient temperature for 0.25 h,then the layers were separated. The organic layer was washed withadditional 0.5 N aqueous HCl, followed by saturated aqueous sodiumchloride (1 L). The organic layer was concentrated under vacuum to givean oil, which later solidified upon standing. The solid could becrystallized from ethyl acetate and hexane to yield methyl[3-bromo-5-formyl-4-(2-methoxyethoxymethoxy)phenyl]-acetate (237 g) as acolorless solid.

Step 2

To a 12-L round bottom flask with a mechanical stirrer, heating mantle,and a reflux condenser was added methyl[3-bromo-5-formyl-4-(2-methoxyethoxymethoxy)-phenyl]acetate (215 g, 595mmol), N-tert-butyl 4-methoxy-5-(benzenesulfonamido)-3-boronic acid(184.5 g, 643 mmol), N,N-diisopropylamine (275 mL, 1.79 mol), and THF (4L). The solution was stirred, degassed under vacuum and placed under anitrogen atmosphere. PdCl₂(dppf).dichloromethane complex (4.86 g, 5.95mmol) was then added in one portion and the solution was heated to 70°C. for 16 h. The reaction mixture was allowed to cool to roomtemperature and it was then concentrated to remove most of the THF (3.5L). The residue was diluted with ethyl acetate (4 L) and then washedwith a 5% solution of potassium carbonate in water (3.0 L), followed bybrine (2.0 L). DARCO-60 charcoal (8 g) was added to the organic phaseand the resulting suspension was stirred at room temperature for 4 h.The solution was then dried over of sodium sulfate (200 g), the organiclayer was then filtered through a fritted filter that was covered inlayers of Celite (300 g), silica gel (300 g), and Celite (300 g). Thefilter cake was washed with a solution of 95:5 dichloromethane/methanol(1 L) and the resulting solution was concentrated in vacuo to give crudemethyl(5′-N-tert-butylsulfamoyl-5-formyl-2′-methoxy-6-methoxyethoxymethoxybiphenyl-3-yl)acetateas an oil, contaminated with methanol (˜12 g) which was taken onto thenext step without additional purification.

Reference E Synthesis of 4-benzyloxy-N-tert-butyl-3-boronicacid-benzenesulfonamide

Method A Step 1

To a 1 L round bottom flask was added 2-iodophenol (50 g) andnitromethane (250 mL) and the reaction mixture was cooled to 0° C.Fuming sulfuric acid (42 mL, 30% SO₃) was added dropwise and thereaction mixture was allowed to warm to room temperature. After 2 h, thereaction was complete and it was poured into water (400 mL) and washedwith ethyl acetate (200 mL). The organic layer was then back extractedwith water (300 mL) and concentrated to oil and combined with theoriginal aqueous layer. The aqueous layer was then neutralized with 5 Maqueous sodium hydroxide (300 mL) and transferred to a 2 L round bottomflask. Sodium hydroxide pellet (11 g), ethanol (150 mL), and benzylbromide (50 mL) were then added and the reaction mixture was heated toan oil bath temperature of 82° C. and stirred for 16 h. After thereaction was complete, ethanol was removed by vacuum distillation whichcaused the product to precipitate out of solution. The product was thenfiltered and dried under high vacuum to give4-benzyloxy-3-iodo-benzenesulfonic acid (61 g, 70% yield).

Step 2

To a 2 L round bottom flask was added 4-benzyloxy-3-iodo-benzenesulfonicacid (49.87 g) and dichloromethane (1000 mL). The suspension was stirredand phosphorous pentachloride (53 g) was added causing the reaction tobecame a solution. After heating the reaction mixture at 40° C. for 1 h,aqueous sodium hydroxide (400 mL of 20%) was then slowly added andstirring was continued until the aqueous was pH 7. The organic layer wasseparated and stirred with 50% aqueous saturated sodium bicarbonate (125mL) for 30 minutes (pH 10). The organic layer was separated, dried withanhydrous sodium sulfate, decanted to a 2 L round bottom flask andtert-butylamine (34 mL) was added. After 16 h, the reaction mixture wasbasified to pH 13-14 with 5% aqueous sodium hydroxide. The organic layerwas separated and concentrated to a solid which was then slurried at 50°C. in isopropyl acetate, cooled, and filtered to give4-benzyloxy-N-tert-butyl-3-iodo-benzenesulfonamide (46 g, 80% yield) intwo crops.

Step 3

To a 1 L round bottom flask was added4-benzyloxy-N-tert-butyl-3-iodo-benzenesulfonamide (32 g) anddichloromethane (320 mL) and the reaction mixture was stirred and cooledto −20 to −25° C. Methyl magnesium bromide (24.4 mL, 3 M in ether) wasadded dropwise. The reaction mixture was stirred for 2 h and then cooledto −35 to −40° C. Isopropyl magnesium bromide (54 mL of 2.13 M in ether)was added dropwise. Tetrahydrofuran (17 mL) and trimethyl borate (6 mL)were then added precipitating a white solid and raising the internaltemperature of the reaction mixture to 0° C. The reaction mixture wasallowed to warm to room temperature and after 12 h phosphoric acid (250mL of 1M in 500 mL of water) was added. The organic layer was separatedand basified with 2.5% aqueous sodium hydroxide (500 mL) causing some ofthe product to precipitate. The aqueous layer along with some of theprecipitated solids was then acidified with concentrated phosphoric acidto a pH of 2 and extracted with 10% tetrahydrofuran in dichloromethane.The solids were carried on with the organic which was then concentratedto give a white solid that was then slurried in 1 L of water for 30minutes. The solid was filtered and dried under high vacuum to give4-benzyloxy-N-tert-butyl-3-boronic acid-benzenesulfonamide (23 g, 88%yield).

Method B: Alternate Synthesis of the Title Compound Step 1

A 3-neck, 3 L-round-bottom flask was equipped with an over-head stirrer,thermometer, N₂ line, 250 mL pressure-equalizing dropping funnel, andgas-exit scrubber to a NaOH solution. The flask was flushed with N₂ andcharged with 2-iodophenol (Alfa Aesar; 201.95, 0.918 mol) and drydichloromethane (920 mL). A gentle stream of N₂ was established throughthe reaction head-space, the reaction vessel then immersed in abrine-ice bath and cooled to −5° C. The dropping funnel was charged withdry dichloromethane (175 mL), then chlorosulfonic acid (Aldrich; 106.96g, 0.918 mol, 1.00 eq.), and the resulting mixture was stirred with aTeflon rod. The dilute solution of chlorosulfonic acid was then addeddropwise to the reaction mixture over a period of approx. 90 mins. Athick pink slurry formed during the addition. Thirty minutes aftercomplete addition, the ice bath was removed and the reaction mixture wasallowed to stir at ambient temperature. After 2 h, the reaction vesselwas immersed in a cold-water bath and water (500 mL) was added to thereaction mixture over a few minutes. The resulting mixture was stirredvigorously until it was biphasic/homogenous upon settling. The mixturewas transferred to a separating funnel along with water and wasextracted with dichloromethane. The aqueous layer containing4-hydroxy-3-iodo-benzenesulfonic acid was transferred back to theoriginal reaction vessel for the next step.

Step 2

Sodium hydroxide (pellets, 110 g, 2.75 mol, 3.00 eq) was addedportionwise to the vigorously stirring aqueous solution of the4-hydroxy-3-iodo-benzenesulfonic acid. After addition was complete,10-15 min., isopropyl alcohol (150 mL) was added to the resulting whitesuspension. The dropping-funnel was charged with benzyl bromide(Aldrich; 164.9 g, 0.964 mol, 1.05 eq.) and added to the reactionmixture over a period of approx. 5 mins. and the reaction mixture washeated to 80°≦T_(int)≦84° C. After approx. 25 min. it was determinedthat the reaction was not proceeding further and therefore additionalsodium hydroxide (3.67 g, 91.8 mmol, 0.1 eq.) and then benzyl bromide(15.7 g, 91.8 mmol, 0.1 eq.) were added to the reaction mixture to givea homogenous solution. After 70 min. from the original benzyl bromideaddition, the heating was stopped and the reaction was allowed to coolslowly in the oil-bath with stirring. At 7.5 h, the reaction mixtureappeared as a suspension of fine-reflective precipitate in brown liquid.The reaction mixture was acidified with 3:1 water-sulfuric acid fromabout pH 13 to between pH 7.5 and 8 (approx. 70 mL is required). Thereaction mixture was then cooled gradually to about 5° C. and stirred atthat temperature for ˜1 h. The waxy white plaques were collected byfiltration, washed with dichloromethane and dried under high vaccum(lyophilizer, 100-200 mTorr) for ˜24 h to give sodium4-benzyloxy-3-iodo-benzenesulfonate as a brilliant white, crystallinesolid, (267.7 g, 71%).

Step 3

A 3-neck, 3 L, round-bottom flask was equipped with an over-headstirrer, reflux condenser (with gas exit to NaOH scrub solution), and apressure-equalizing dropping-funnel with N₂ line. The flask was flushedwith N₂, charged with sodium 4-benzyloxy-3-iodo-benzenesulfonate (234 g,0.568 mol), dichloromethane (1.15 L), and catalytic amount ofdimethylformamide (910 mg, 11.7 mmol, 2.1 mol %). The white suspensionwas stirred under a gentle stream of nitrogen and heated in an oil-bathset between 40 and 45° C. Oxalyl chloride (90.1 g, 0.710 mol, 1.25 eq)was then added over 3-5 min. After 2.5 h, the reaction was allowed tocool to 25° C. in a cold-water bath and then quenched drop-wise withwater (60 mL) over approx. 5 min. A further portion of water (450 mL)was added in a single portion and the reaction mixture stirredvigorously for 5 to 10 min. The organic layer was separated and washedwith water until the aqueous pH had increased to pH 4 to 5). Theresulting dichloromethane solution of 4-benzyloxy-3-iodo-benzenesulfonylchloride was used in the next step.

Step 4

A 3-neck, 3 L, round-bottom flask was equipped with an over-headstirrer, thermometer, and a pressure-equalizing dropping-funnel wascharged with the solution of 4-benzyloxy-3-iodo-benzenesulfonylchloride. The flask immersed in a cold water bath (T_(int)=22° C.) andtert-butylamine (90.1 g, 0.710 mol, 2.1 eq) was added drop-wise (T_(int)no change). The resulting reaction mixture was stirred overnight at theambient water-bath temperature. After 17 h, the reaction mixture wasworked-up and the organic layer was separated and concentrated toapprox. ⅓ (˜500 mL) of its original volume at which point the productstarted to precipitate. The reaction mixture was warmed to 35-40° C. atatmospheric pressure till the solids had re-dissolved. The solution wasthen allowed to cool, with gentle stirring, to room temperature. Within2 days a white precipitate had formed. The suspension was stirredvigorously while hexane (1.5 L) was slowly added, then stirredovernight, and then cooled in an ice-bath for 1-2 h The precipitate wascollected by filtration and washed with hexane, dried, first undersuction to give 4-benzyloxy-N-tert-butyl-3-iodo-benzenesulfonamide (238,94%).

Step 5

A 3-neck, 2 L-round-bottom flask was equipped with an over-head stirrer,thermometer, pressure-equalizing dropping-funnel, and an N₂ line. Theflask was flushed with N₂ and then charged with4-benzyloxy-N-tert-butyl-3-iodo-benzenesulfonamide (198.6 g, 0.446 mol)and dichloromethane (600 mL). The white suspension was stirred under agentle stream of N₂ and cooled in an ice-water bath (0° C.≦T_(int)≦5°C.). The dropping-funnel was charged with methyl magnesium bromide(Aldrich; 3.0 M in diethyl ether, 167 g, ˜171 mL, 0.513 mol, 1.15 eq),which was added dropwise to the suspension at such a rate so as tomaintain T_(int)<5° C. (addition of salt to the cool-bath was necessary)to give a colorless-homogenous mixture within ˜⅓ addition. After theaddition was completer, the dropping-funnel was charged withisopropylmagnesium bromide (Boulder Scientific; 2.13 M in diethyl ether;250 mL, 0.533 mol, 1.2 eq), which was added dropwise to the reactionmixture at such a rate so as to maintain T_(int)<5° C. After theaddition was complete, the reaction mixture was stirred for 15-20 min.The dropping-funnel was removed and replaced with a septa and cannula,and the reaction mixture was transferred over 2 h to a 3-necked, 3-Lround bottom flask containing a solution trimethyl borate (106.6 g, 1.03mol, 2.30 eq) and tetrahydrofuran (600 mL) and maintained under nitrogenatmosphere at (T_(int)<5° C.) utilizing an ice-water bath. After theaddition was complete, the solution was allowed to stir at <5° C. for 30min. and then transferred to a separatory funnel and washed with anequal volume of a 2:1 (water:phosphoric acid) solution. The organiclayer was dried over sodium sulfate. Ethyl acetate was added to thesolution and the combined organic layer was concentrated to give4-benzyloxy-N-tert-butyl-3-boronic acid-benzenesulfonamide (129 g, 80%).

Reference F Synthesis of (4-benzyloxy-3-bromo-5-formyl-phenyl) aceticacid methyl ester

To a 250 mL round-bottomed flask with a teflon stir bar and a refluxcondensor were added methyl (3-bromo-5-formyl-4-hydroxyphenyl)acetate(23.3 g, 85.3 mmol, prepared as described in Reference A above),dichloromethane (100 mL), benzyl bromide (15.32 g, 10.64 mL, 89.59mmol), and N-ethyldiisopropylamine (11.6 g, 15.6 mL, 89.5 mmol). Thereaction mixture was heated to reflux for 14 h and then allowed to coolto room temperature. Dichloromethane (100 mL) was added and the organiclayer was separated and washed with 200 mL of a 3% solution of NaOH inwater. The organic layer was concentrated to give(4-benzyloxy-3-bromo-5-formyl-phenyl) acetic acid methyl ester (30.65 g)as oil.

Example 1 Synthesis of(S)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinicacid

Method A Step 1

To a 12-L round bottom flask with stir bar was added methyl(5′-N-tert-butylsulfamoyl-5-formyl-2′-methoxy-6-methoxyethoxymethoxybiphenyl-3-yl)acetate(324.3 g), methanol (5.2 L), followed by addition of concentratedaqueous HCl (0.29 L) dropwise to maintain the internal temperature ofthe flask below 25° C. The reaction mixture was allowed to stir at roomtemperature overnight by which time the product had precipitated fromsolution. The reaction mixture was cooled to −20° C., and filtered andthe resulting solid was then washed with an additional portion ofmethanol (750 mL) that had been previously cooled to −25° C. Theoff-white solid was dried in a vacuum chamber to give methyl[5′-N-tert-butylsulfamoyl-5-formyl-6-hydroxy-2′-methoxy)biphenyl-3-yl]acetate(205 g).

Step 2

To a 12-L round bottom flask with a mechanical stirrer and heatingmantle was added methyl[5′-N-tert-butylsulfamoyl-5-formyl-6-hydroxy-2′-methoxy)biphenyl-3-yl]acetate(200.8 g, 461 mmol), isopropanol (2.3 L) and a solution of sodiummetabisulfite (106.4 g, 553 mmol) dissolved in water (0.21 L). Theresulting suspension was heated to 60° C. for 2.5 h.3,4-Diaminobenzamidine monohydrochloride (115.2 g, 617 mmol) was addedand a stream of air was then allowed to circulate through the flask. Thesolution turned orange and became homogeneous after 0.25 h. After 1 h,the reaction was shown to be 70% complete by HPLC analysis, but requireda reaction time of about 18 h to go to completion resulting in theprecipitation of the product. The reaction mixture was cooled to roomtemperature and filtered. The solid was washed with isopropanol (1 L)and dried in a vacuum chamber to give methyl[5′-N-tert-butylsulfamoyl-5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6-hydroxy-2′-methoxybiphenyl-3-yl]-acetate(264 g) as a mustard-colored solid.

Step 3

To a 3-L round bottom flask was added methyl[5′-N-tert-butylsulfamoyl-5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6-hydroxy-2′-methoxybiphenyl-3-yl]-acetate(49.9 g, 82.9 mmol) and trifluoroacetic acid (770 mL). The reactionmixture was allowed to stir at room temperature for 1.5 h thenconcentrated in vacuo. Methanol (1 L) was added to the gel-like residueand the solution was concentrated again then dried under high vacuumovernight. The dry, solid product was transferred to a 3 L 3-neck roundbottom flask fitted with a Dean-Stark trap, a stirring shaft and anitrogen inlet. The flask was flushed with nitrogen then pyridinehydrochloride (412 g, 3.57 mol) and toluene (100 mL) were added. Thereaction mixture was heated under reflux to remove residual water fromthe pyridine salt and solvent. The solution was heated to 150° C. tomelt the pyridine hydrochloride after which the temperature wasincreased to 180° C. over 0.5 h. After 1.5 h, the solution was cooledunder a positive pressure of nitrogen until it solidified and reachedroom temperature. Water (3 L) was added and the solution was allowed tostir at room temperature overnight. The reaction mixture was filteredand the solid was washed with water (200 mL) and isopropanol (100 mL).The solid was dried in a vacuum chamber overnight to yield[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoyl-biphenyl-3-yl]-aceticacid (38.15 g) which was contaminated with about 4% sulfonic acid.

Alternative synthesis of[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoyl-biphenyl-3-yl]-aceticacid

Pyridine (69.0 g, 70.4 mL, 0.87 mol) in anhydrous ether (200 mL) wastreated with 4 M HCl in dioxane (250 mL, 1.0 mol) until precipitation ofthe salt was complete. Excess solvent was removed by evaporation andtoluene (500 mL) was added. The reaction mixture was refluxed for 2-3 hwith a Dean-Stark trap. After cooling, toluene was evaporated ascompletely as possible and the flask containing the solid pyridine.HClwas immersed in an oil bath heated at 180-185° C. and allowed to melt.The melted pyridine.HCl was heated at 180-185° C. for 30-60 minuteswhile connected to a drying tube. Next, the melted salt was treated inone portion with moderate stirring with methyl[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6-hydroxy-2′-methoxy-5′-sulfamoyl-biphenyl-3-yl]acetate(25.0 g, 29.4 mmol) (obtained from the TFA treatment of methyl[5′-N-tert-butylsulfamoyl-5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6-hydroxy-2′-methoxybiphenyl-3-yl]-acetate).The hot solution was heated with stirring for 60-90 minutes until thereaction was complete by HPLC analysis. After cooling, the solid mixturewas dissolved in water (800 mL) and stirred overnight. The resultingsolid was filtered and dried in vacuo to give[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoyl-biphenyl-3-yl]-aceticacid (13-14 g) containing ˜1-2% of sulfonic acid impurities.

Step 4

To a magnetically stirred solution of[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoyl-biphenyl-3-yl]-aceticacid (14.0 g, 27.1 mmol) in N,N-dimethylacetamide (125 mL) at 0° C. wasadded N-methylmorpholine (5.95 mL, 54.2 mmol), EDC (5.71 g, 29.8 mmol)and HOBt (4.00 g, 29.8 mmol). The reaction mixture was allowed to stirat 0° C. for 0.5 h. A solution of H-Asp-(OBn)-OBn.HCl (10.4 g, 29.8mmol) in DMA (25 mL) was added to the reaction mixture and the resultingsolution was stirred at ambient temperature for 12 h. The solution wasconcentrated in vacuo under high vacuum, maintaining the water bathtemperature below 50° C. to remove most of the DMA. The residual oil wassuspended in acetonitrile (150 mL) and the reaction mixture was allowedto stir at ambient temperature for 0.5 h, after which time the producthad precipitated from solution. The solid was removed by filtration andit was washed with acetonitrile. The solid was suspended in a saturatedaqueous solution of sodium bicarbonate (150 mL), stirred at roomtemperature for 0.5 h, and then filtered. The resulting solid was thensuspended in a 10 mM aqueous HCl solution (150 mL) and allowed to stirat room temperature for 0.5 h. The suspension was filtered and dried togive dibenzyl(S)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinate(18 g) that was 80-90% pure by analytical HPLC. The crude dibenzyl(S)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinatewas taken directly onto the next step without additional purification.

Step 5

Crude dibenzyl(S)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinate(18 g) was dissolved in a 1:1 mixture of acetic acid and water (800 mL),assisted by sonication. To this solution was added palladium hydroxideon carbon (Pearlman's catalyst, 20 wt % Pd on a dry weight basis,containing 50% water by weight; 7.0 g). The reaction mixture waspressurized to 30 psi with hydrogen and the suspension was shaken on aParr apparatus for 10 h. The catalyst was removed by filtration and thesolvent was removed in vacuo. Crude(S)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinicacid was purified by preparative reverse-phase HPLC to give(S)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinicacid (7.2 g).

Method B: Alternate Synthesis of[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoyl-biphenyl-3-yl]-aceticacid Step 1

To a 1 L round bottom flask with magnetic stir bar and a nitrogen refluxcondensor was added crude (4-benzyloxy-3-bromo-5-formyl-phenyl) aceticacid methyl ester (19.36 g, 53.31 mmol), tetrahydrofuran (400 mL),diispropyl amine (16.7 g, 23.2 mL, 165.1 mmol) and4-benzyloxy-N-tert-butyl-3-boronic acid-benzenesulfonamide (21.74 g,59.7 mmol). The reaction mixture was placed under a nitrogen atmosphereanddichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)dichloromethaneadduct (0.68 g, 0.832 mmol) was added. The reaction mixture was heatedto reflux for 16 h at which time it was complete by HPLC. The solutionwas concentrated to a solid and methyl tert-butyl ether (500 ml) wasadded to dissolve the solid. The solution was washed twice with 3% coldaqueous sodium hydroxide solution (500 mL). The organic layer wasfiltered through a plug of Celite (200 g) and silica gel (100 g). TheCelite was washed with a 1:1 mixture of methyl tert-butyl ether andethyl acetate (400 mL). The filterate was concentrated to give(6,2′-bisbenzyloxy-5′-N-tert-butylsulfamoyl-5-formyl-biphenyl-3-yl)-aceticacid methyl ester (32 g) as oil, which was taken directly onto the nextstep without additional purification.

Step 2

To a 250 mL round-bottom flask with stir bar was added crude(6,2′-bisbenzyloxy-5′-N-tert-butylsulfamoyl-5-formyl-biphenyl-3-yl)-aceticacid methyl ester (5.00 g, 8.31 mmol), methanol (50 mL) and water (5mL). The reaction mixture was warmed to 60° C. and sodium metabisulfite(1.58 g, 8.31 mmol) was added, and the reaction mixture was allowed tostir at 60° C. for 2.5 h. 3,4-Diaminobenzamidine hydrochloride (1.80 g,9.64 mmol) was added in one portion and the reaction mixture was heatedat 60° C. overnight. The reaction mixture was cooled to room temperatureand 250 mL of a (3:1) isopropyl acetate:isopropanol solution was addedfollowed by addition of water (50 mL). The organic layer was separated,filtered through Celite, dried over sodium sulfate, and concentrated togive[6,2′-bisbenzyloxy-5′-N-tert-butylsulfamoyl-5-(5-carbamimidoyl-1H-benzimidazolyl-2-yl)-biphenyl-3-yl]-aceticacid methyl ester hydrochloride (5.8 g, 91%) as a light brown solid.

Alternative Method to Step 2

To a 500 mL round bottom flask with stir bar and a reflux condensor wasadded crude(6,2′-bisbenzyloxy-5′-N-tert-butylsulfamoyl-5-formyl-biphenyl-3-yl)-aceticacid methyl ester (20 g, 33.23 mmol) and 3,4-diaminobenzamidine (7.44 g,39.88 mmol), PdCl₂(MeCN)₂ (100 mg, Strem Chemicals), followed byaddition of a 9:1 mixture of isopropanol:water (200 mL) and a solutionof sodium metabisulfite (6.3 g, 33.2 mmol) in water (30 mL). Thereaction mixture was heated to reflux with an oil bath and the top ofthe reflux condensor was left open to the air. The solution was heatedto reflux overnight, at which time it was complete by HPLC. To thissolution was added Darco (5 g) and the solution was filtered hot throughcelite (100 g). The celite was washed with isopropanol (100 mL) and theresulting solution was extracted with ethyl acetate (300 mL) and brine(70 mL). The organic layer was concentrated to give[6,2′-bisbenzyloxy-5′-N-tert-butylsulfamoyl-5-(5-carbamimidoyl-1H-benzimidazolyl-2-yl)biphenyl-3-yl]-aceticacid methyl ester hydrochloride as an amorphous solid that was carriedonto the next step without any additional purification.

Step 3

To a 50 mL round bottom flask was added[6,2′-bisbenzyloxy-5′-N-tert-butylsulfamoyl-5-(5-carbamimidoyl-1H-benzimidazolyl-2-yl)-biphenyl-3-yl]-aceticacid methyl ester hydrochloride (0.500 g, 0.651 mmol) and glacial aceticacid (2.5 mL). 12 N HCl (aq) (2.5 mL) was added and the reaction mixturewas heated to 50° C. After 14 h, the reaction mixture was added to water(100 mL) and the solution was concentrated to 5 mL in vacuo to give aprecipate. The precipitate was removed by filtration to give[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoyl-biphenyl-3-yl]-aceticacid (365 mg) which contained water and 2.8 eq. of HCl (93%) which wasthen be converted to the title compound as described above.

Alternative Method to Step 3

To a 1 L round bottom flask was added crude[6,2′-bisbenzyloxy-5′-N-tert-butylsulfamoyl-5-(5-carbamimidoyl-1H-benzimidazolyl-2-yl)-biphenyl-3-yl]-aceticacid methyl ester hydrochloride (22.0 g, 28.65 mmol) and glacial aceticacid (400 mL). To this solution was added 12 N HCl (aq) (400 mL) and thereaction mixture was allowed to heat to 55° C. and the reaction was >95%complete by HPLC after 14 h. To this solution was added Darco (2 g). Thesolution was allowed to stir at 55° C. for an additional hour and thesolution was filtered through celite to remove the charcoal. Thesolution was concentrated under reduced pressure to a solid which wastreated with 1N HCl (600 mL) and acetonitrile (200 mL). Upon heating thesolid dissolved and at that time Darco (6 g) was added. The solution wasallowed to stir at 70° C. for 1 h and it was filtered hot throughcelite. The solution was allowed to cool to room temperature and theprecipitate was filtered to give[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoyl-biphenyl-3-yl]-aceticacid (9.85 g, 66%).

Example 2 Synthesis of(S)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinicacid zwitterion

Purified lyophilized(S)-2-{2-[5-(5-carbamimidoyl-1H-benzimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoylbiphenyl-3-yl]acetylamino}succinicacid (11 g) powder was suspended in 130 mL of 2 M HCl (aqueous) andwarmed to 60° C. THF (80 mL) was added to this suspension and stirred atthat temperature until the compound was completely dissolved (pH of thesolution was kept around ˜2.0). This solution was filtered while hot andstirred slowly at room temperature for 24 h after adding seed crystals.Crystalline material (5.2 g) was filtered at room temperature, waswashed with water until washings were neutral (pH 7). Elemental analysisand chloride content analysis revealed absence of any chloride counterion, indicating that the material is zwitter-ionic.

¹H NMR (DMSO-d₆): 9.21 (2H, br s), 8.87 (2H, br s), 8.21 (1H, d, J=7.8Hz), 8.0 (2H, d, J=7.2 Hz), 7.79 (1H, d, J=7.8 Hz), 7.73 (J=1.5 Hz, 1H,d), 7.6 (J=8.6, 1.5 Hz, dd, 1H), 7.5 (1H, dd, J=8.5 and 2.5 Hz), 7.2(1H, d, J=2.3 Hz), 4.3 (1H, J=2.3, 5.3 Hz, dd), 3.5 (2H, s), 2.6 (1H,dd, J=16 and 7.3 Hz), 2.44 (1H, dd, J=5.3 Hz).

Biological Examples Example 1 In Vitro Factor VIIa Inhibitor Assay

Mixtures of human Factor VIIa (typically supplied at 7 nM) and testcompound (present at varying concentrations) in assay medium(comprising: NaCl, 150 mM (pH 7.4); CaCl₂, 5 mM; Tween-20, 0.05%; DadeInnovin tissue factor [Dade Behring, Newark, Del., USA]; EDTA, 1.5 mM;and dimethylsulfoxide, 10%) were incubated for 30 minutes at roomtemperature. Next, reactions were initiated with the addition ofsubstrate [500 μM of CH₃SO₂-D-Cha-But-Arg-pNA (from Centerchem, Norwalk,Conn., USA)]. Hydrolysis of the chromogenic substrate was followedspectrophotometrically at 405 nm for five minutes. Initial velocitymeasurements calculated from the progress curves by a kinetic analysisprogram (Batch Ki; BioKin, Ltd., Pullman, Wash.) were used to determineapparent inhibition constants (apparent K_(i)'s).

Compounds of the invention tested by the above-described assay exhibitedinhibition of Factor VIIa.

Example 2 In Vitro Factor Xa Inhibitor Assay

Mixtures of human Factor Xa (typically supplied at 3 nM) (fromHaematologic Technologies, Essex Junction, Vt., USA) and test compound(varying concentrations) in assay medium (comprising: Tris, 50 mM (pH7.4); NaCl, 150 mM; CaCl₂, 5 mM; Tween-20, 0.05%; EDTA, 1 mM; anddimethylsulfoxide, 10%) were incubated for 30 minutes at roomtemperature. Next, reactions were initiated with the addition ofsubstrate [500 μM of CH₃CO₂-D-Cha-Gly-Arg-pNA (from Centerchem, Norwalk,Conn., USA]. Hydrolysis of the chromogenic substrate was followedspectrophotometrically at (405 nm) for five minutes. Apparent inhibitionconstants (apparent K_(i)'s) were calculated from the enzyme progresscurves using standard mathematical models.

Compounds of the invention tested by the above-described assay exhibitedinhibition of Factor Xa.

Example 3 Pharmacokinetic Assay

Rats with pre-implanted jugular vein catheters, which were filled withheparin/saline/PVP lock prior to shipment, were bought from CharlesRiver. Three rats were selected for each study, weighed, and injectedwith test compound by tail vein injection. Any residual test compoundwas retained and stored at −70° C. for later analysis.

Blood samples (0.25 mL each) were collected from the indwellingcatheters at specified times over 120 h. The catheters were flushed withphysiological saline immediately after each collection and filled withheparinized saline after each 8, 24 and 48 h collection. In the eventthat a catheter failed, blood samples were collected via theretro-orbital sinus under isoflurane anesthesia at the appropriate time.

Blood samples were placed in 0.5 mL Microtainer® tubes (lithiumheparin), shaken gently and stored on wet ice. The samples werecentrifuged for 10 minutes at 2400 rpm in a refrigerated centrifuged.Plasma samples (0.1 mL) from each tube were transferred to 0.5 mL Unisonpolypropylene vials (Sun—500210) and stored below −70° C. for lateranalysis by LC/MS-MS.

Example 4 In Vitro Clotting Assays . . . aPTT and PT

Coagulation assays, activated partial thromboplastin time (aPTT) andprothrombin time (PT) were carried out based on the procedure describedin Hougie, C. Hematology (Williams, W. J., Beutler, B., Erslev, A. J.,and Lichtman, M. A., Eds.), pp. 1766-1770 (1990), McGraw-Hill, New York.

Briefly, the assays were performed using normal human citrated plasmaand were performed at 37° C. on a coagulometer (Electra 800) inaccordance with the manufacturer's instructions (Medical LaboratoryAutomation—Pleasantville, N.Y.). The instrument was calibrated withplasma immediately prior to collecting clotting times for samples withinhibitors. The aPTT and PT doubling concentrations were calculated byfitting inhibitor dose response curves to a modified version of the Hillequation.

Pharmaceutical Composition Examples

The following are representative pharmaceutical formulations containinga compound of Formula I.

Tablet Formulation

The following ingredients are mixed intimately and pressed into singlescored tablets.

Quantity per Ingredient tablet, mg compound of this invention 400cornstarch 50 croscarmellose sodium 25 lactose 120 magnesium stearate 5

Capsule Formulation

The following ingredients are mixed intimately and loaded into ahard-shell gelatin capsule.

Quantity per Ingredient capsule, mg compound of this invention 200lactose, spray-dried 148 magnesium stearate 2

Suspension Formulation

The following ingredients are mixed to form a suspension for oraladministration.

Ingredient Amount compound of this invention 1.0 g fumaric acid 0.5 gsodium chloride 2.0 g methyl paraben 0.15 g propyl paraben 0.05 ggranulated sugar 25.5 g sorbitol (70% solution) 12.85 g Veegum K(Vanderbilt Co.) 1.0 g flavoring 0.035 mL colorings 0.5 mg distilledwater q.s. to 100 mL

Injectable Formulation

The following ingredients are mixed to form an injectable formulation.

Ingredient Amount compound of this invention 1.2 g sodium acetate buffersolution, 0.4M 2.0 mL HCl (1N) or NaOH (1N) q.s. to suitable pH water(distilled, sterile) q.s. to 20 mL

All of the above ingredients, except water, are combined and heated to60-70° C. with stirring. A sufficient quantity of water at 60° C. isthen added with vigorous stirring to emulsify the ingredients, and waterthen added q.s. to 100 g.

Suppository Formulation

A suppository of total weight 2.5 g is prepared by mixing the compoundof the invention with Witepsol® H-15 (triglycerides of saturatedvegetable fatty acid; Riches-Nelson, Inc., New York), and has thefollowing composition:

compound of the invention 500 mg Witepsol ® H-15 balance

Parenteral Formulation

Compound (Ib)  40 mg/mL Hydroxypropyl-β-cyclodextrin 200 mg/mL

-   -   Adjust pH with 1.0 N sodium hydroxide to 7.4

The foregoing invention has been described in some detail by way ofillustration and example, for purposes of clarity and understanding. Itwill be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

We claim:
 1. A method of effecting clotting in an individual in needthereof, comprising administering to the individual an isolated compoundof Formula Ib:

or a zwitterion thereof or a pharmaceutically acceptable salt thereof;substantially free of the opposite enantiomer of the compound of FormulaIb.
 2. A method of effecting clotting in an individual in need thereof,comprising administering to the individual a pharmaceutical compositioncomprising: a. an isolated compound of Formula Ib:

or a zwitterion thereof or a pharmaceutically acceptable salt thereof;substantially free of the opposite enantiomer of the compound of FormulaIb; and b. a pharmaceutically acceptable carrier or excipient.
 3. Themethod of claim 2, wherein the pharmaceutical composition is formulatedfor parenteral administration.
 4. The method of claim 2, wherein thepharmaceutical composition is formulated for intravenous or subcutaneousadministration.
 5. The method of claim 2, wherein the pharmaceuticalcomposition further comprises an additional active ingredient.
 6. Themethod of claim 2, wherein the pharmaceutical composition compriseswater.
 7. The method of claim 2, wherein the pH of the pharmaceuticalcomposition is adjusted to a pharmaceutically acceptable pH.